Neurally evoked responses of human isolated resistance arteries are mediated by both alpha 1- and alpha 2-adrenoceptors. 1. Human subcutaneous resistance arteries (internal diameter 113-626 microns) were mounted in an isometric myograph. Electrical field stimulation was applied either continuously in the form of a frequency-response curve or intermittently at 16 Hz. The magnitude of the maximum contraction induced by continuous stimulation expressed as a percentage of the response to a supramaximal concentration of noradrenaline (10 microM) was highly variable but unrelated to vessel calibre. Contractile responses to both continuous and intermittent stimulation were abolished by 1 microM tetrodotoxin. 2. Prazosin (100 nM and 1 microM, alpha 1-adrenoceptor antagonist) inhibited responses to continuous stimulation over a range of frequencies (2-8 Hz). The response to continuous stimulation at 8 Hz was inhibited by 78 +/- 6% by 1 microM prazosin. Rauwolscine (100 nM, alpha 2-adrenoceptor antagonist) had a smaller effect on contractions induced by continuous stimulation. Rauwolscine inhibited the response at 8 Hz by 36 +/- 6%. Rauwolscine at a higher concentration (1 microM) caused further inhibition of the response to continuous stimulation. Prazosin and rauwolscine in combination almost completely inhibited the response to continuous stimulation at concentrations of 1 microM. 3. Prazosin and rauwolscine inhibited responses to intermittent stimulation in a concentration-dependent manner. The IC50 for this action of prazosin was 3.7 +/- 1.6 nM and the maximum inhibition induced by 100 nM prazosin was 78 +/- 6%. The IC50 of rauwolscine was 12.0 +/- 1.3 nM and 100 nM rauwolscine caused a 86 +/- 7% reduction in the response to intermittent stimulation.Prazosin and rauwolscine in combination (each at 100 nM) caused marked inhibition of the response to intermittent stimulation leaving only 7.0 +/- 2.6% of the response.4. These data suggest that neurally released noradrenaline evokes contractions of human resistance arteries by activation of both alpha 1,- and alpha 2-adrenoceptors postjunctionally.


Calcium-activated currents in cultured neurones from rat dorsal root ganglia. 1. Voltage-activated Ca2+ currents and caffeine (1 to 10 mM) were used to increase intracellular Ca2+ in rat cultured dorsal root ganglia (DRG) neurones. Elevation of intracellular Ca2+ resulted in activation of inward currents which were attenuated by increasing the Ca2+ buffering capacity of cells by raising the concentration of EGTA in the patch solution to 10 mM. Low and high voltage-activated Ca2+ currents gave rise to Cl- tail currents in cells loaded with CsCl patch solution. Outward Ca2+ channel currents activated at very depolarized potentials (Vc + 60 mV to + 100 mV) also activated Cl- tail currents, which were enhanced when extracellular Ca2+ was elevated from 2 mM to 4 mM. 2. The Ca(2+)-activated Cl- tail currents were identified by estimation of tail current reversal potential by use of a double pulse protocol and by sensitivity to the Cl- channel blocker 5-nitro 2-(3-phenyl-propylamino) benzoic acid (NPPB) applied at a concentration of 10 microM. 3. Cells loaded with Cs acetate patch solution and bathed in medium containing 4 mM Ca2+ also had prolonged Ca(2+)-dependent tail currents, however these smaller tail currents were insensitive to NPPB. Release of Ca2+ from intracellular stores by caffeine gave rise to sustained inward currents in cells loaded with Cs acetate. Both Ca(2+)-activated tail currents and caffeine-induced inward currents recorded from cells loaded with Cs acetate were attenuated by Tris based recording media, and had reversal potentials positive to 0 mV suggesting activity of Ca(2+)-activated cation channels.4. Our data may reflect (a) different degrees of association between Ca2+-activated channels with voltage-gated Ca2+ channels, (b) distinct relationships between channels and intracellular Ca2" stores and Ca2+ homeostatic mechanisms, (c) regulation of Ca2+-activated channels by second messengers, and (d) varying channel sensitivity to Ca2 , in the cell body of DRG neurones.


The selective action of quinacrine on high-threshold calcium channels in rat hippocampal cells. 1. The whole-cell patch-clamp technique has been used to examine Ca channel currents carried by Ba (IBa) in rat hippocampal neurones. 2. Quinacrine selectivity decreased the high-threshold current activated by membrane depolarization from a holding potential of -70 mV. Neither the low-threshold Ca channel current nor the fast tetrodotoxin (TTX)-sensitive sodium current were affected by quinacrine. 3. Bath application of quinacrine caused a dose-dependent reduction of the peak amplitude of IBa. This effect was fast, voltage-independent, reversible and had a Kd of 30 +/- 5 microM. 4. The quinacrine-induced block did not change the time-course and the voltage dependence of IBa activation and deactivation. The inhibition revealed no use-dependence, ruling out an open channel block by quinacrine. 5. p-Bromophenacyl bromide had no effect on IBa suggesting the lack of involvement of phospholipase A2 in the action of quinacrine. In addition, the quinacrine-induced block was not related to the calmodulin pathway and internal quinacrine did not affect the peak amplitude of IBa. 6. The effect of quinacrine on the amplitude of IBa was dependent of the external pH, and suggested that only the single-protonated form of the drug can bind to the channel receptor with a Kd of 3 microM. Quinacrine and other substituted acridines can thus be useful for pharmacological and structure-activity studies of Ca channels.


Osmotic regulation of neuropeptide Y and its binding sites in the magnocellular hypothalamo-neurohypophysial pathway. The magnocellular hypothalamo-neurohypophysial system is, via a release of vasopressin from nerve terminals in the neurohypophysis to the peripheral blood, centrally involved in the regulation of body salt and water homeostasis. Furthermore, it has been shown that expression of neuropeptides co-existing with vasopressin or oxytocin in magnocellular neurons is influenced by salt loading. We here report, that neuropeptide Y (NPY)-immunoreactivity, which is normally not observed in the magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei of rats becomes immunohistochemically detectable after salt loading. Using a double-immunohistochemical procedure on the same brain sections, it is shown that NPY is co-existing with either vasopressin or oxytocin in these neurons. Within the neurohypophysis of normal rats, a moderate number of predominantly fine calibered NPY-immunoreactive nerve fibers most often coursing along vessels is observed in addition to a low number of large peptidergic terminals. In salt-loaded rats, however, the number of NPY-immunoreactive neurohypophysial large nerve terminals in apposition to vascular lumina is drastically increased. By using quantitative receptor autoradiography, it is demonstrated that in salt-loaded animals, the number of neurohypophysial NPY binding sites is decreased to nearly undetectable levels (0.054 +/- 0.02 fmol/mg) compared to a very high density of binding sites in normal animals (1.151 +/- 0.15 fmol/mg). This raises evidence that NPY containing hypothalamo-neurohypophysial neurons as well as peripherally released NPY may be involved in the regulation of water homeostasis via NPY receptors in the neurohypophysis.


Chronic treatment with a benzodiazepine agonist in vivo increases the actions of the benzodiazepine partial inverse agonist, FG7142, on the hippocampal slice in vitro. We have shown previously that chronic treatment of mice with a benzodiazepine agonist, flurazepam, increased the pharmacological actions of the partial inverse agonist, FG7142. We have investigated the neurophysiological basis for this using extracellular recordings of evoked field potentials in area CA1 of isolated hippocampal slices. The slices were prepared 48 h after the end of the chronic in vivo treatment, a time when no evidence of residual benzodiazepine agonist activity was found in the CNS. During perfusion with standard Ringer solution, no significant differences were seen between the field potentials in slices from flurazepam-treated mice and those from control animals. When FG7142 was added to the perfusion medium there was an increase in the secondary discharges that followed the initial population spikes, and an increase in paired pulse potentiation. These increases were significantly greater in slices from flurazepam-treated mice, compared with controls. The results show that the effects of the partial inverse agonist, FG7142, on an isolated neuronal preparation, were increased by chronic administration of a benzodiazepine agonist in vivo. This effect is suggested to be due to a decrease in GABAergic inhibition.


GABAergic influence on the development of the sexually dimorphic nucleus of male and female rats. A large number of estrogen-sensitive neurons in rat hypothalamus use gamma-aminobutyric acid (GABA) as a neurotransmitter. As estrogens influence the size of the sexually dimorphic nucleus (SDN) of the preoptic area it was tested whether perinatal treatment of rats with the GABA-agonistic drug muscimol can induce similar changes as estrogens. Male and female rats were treated perinatally with muscimol or with the vehicle only and the vols. of the SDN of the preoptic area were determined morphometrically following maturation of the animals. The SDN vols. in treated males were significantly smaller (80.3%) compared to controls. There was no striking effect in females. Our data suggest an influence of the GABAergic system on the development of the sexually dimorphic nucleus of the preoptic area (SDN-POA). The muscimol treatment, however, did not mimic the effects of perinatal estrogen treatment. Since muscimol is a GABAA receptor stimulating drug it appears that this receptor subtype is not involved in the estrogen-induced changes in size of the SDN.


Cyclic AMP analog activates Na(+)-dependent inward currents in dissociated frog motoneurons. Effects of intracellular accumulation of 3',5'-cyclic adenosine monophosphate (cAMP) were studied using 3',5'-cyclic 8-bromoadenosine monophosphate (8-Br-cAMP) and forskolin on single motoneurons acutely dissociated from adult bullfrog spinal cord. 8-Br-cAMP (10(-3) M) and forskolin (1.5 x 10(-6) M) activated inward currents under K(+)-free conditions at a holding potential of -70 mV. The currents were dependent on extracellular Na+ concentration, and were never reversed within the range of membrane potentials tested (-130 to 30 mV). These results indicate that accumulation of intracellular cAMP induces Na(+)-dependent inward currents in frog motoneurons.


An inward rectifier is present in presynaptic nerve terminals in the chick ciliary ganglion. Inwardly rectifying voltage-sensitive channels have been detected in the cell bodies and axons of a number of excitable cells. The question of whether similar channels exist at axon terminals has been a matter of speculation for some time. We now report the first direct evidence for the existence of inward rectifiers in vertebrate presynaptic nerve terminals. Following impalement with intracellular electrodes, the large calyciform nerve terminals innervating chick ciliary ganglion neurons exhibit pronounced inward rectification upon hyperpolarization that increases with increasing current strength. The response is blocked by 2 mM Cs+, but is insensitive to Ba2+, tetraethylammonium and tetrodotoxin. The inward rectifier exhibits dependence on both Na+ and K+, but is unaffected by altering extracellular Ca2+. Ciliary neurons innervated by these nerve terminals display inward rectification with similar properties. We conclude that the inward rectifier present in these presynaptic nerve terminals resembles the H-current previously described in sensory ganglion neurons and the Q-current found in hippocampal pyramidal neurons. The presence of channels that are activated by hyperpolarization may serve to enhance the excitability of the calyciform nerve terminals, which are capable of relatively high frequencies (greater than 100 Hz) of discharge.


Binding sites for pituitary adenylate cyclase activating polypeptide (PACAP): comparison with vasoactive intestinal polypeptide (VIP) binding site localization in rat brain sections. Pituitary adenylate cyclase activating polypeptide (PACAP) is structurally similar to vasoactive intestinal polypeptide (VIP). We investigated the characteristics and topographical distribution of [125I]PACAP binding sites compared with those of [125I]VIP binding sites in the rat brain. Radiolabeled PACAP and VIP showed highly specific binding to sections at the level of the dorsal hippocampus. The specific binding of [125I]PACAP was 10 times higher than that of [125I]VIP in hippocampal sections. [125I]PACAP binding was scarcely displaced by unlabeled VIP, while [125I]VIP binding was effectively displaced by unlabeled PACAP. Therefore, PACAP binding sites may reflect both PACAP specific binding sites and VIP/PACAP binding sites. However, the amount of VIP/PACAP binding sites was negligibly low. Autoradiography revealed that [125I]PACAP binding sites were dense in the piriform cortex, diagonal band, accumbens nucleus, anterior part of the striatum, hippocampal formation, habenular nucleus, lateral hypothalamic area, superior colliculus and dorsal raphe nucleus. Moderate to high labeling was observed in the medial septal nucleus, olfactory tubercle, caudal part of the striatum, most parts of the thalamus, supraoptic and periventricular hypothalamic nuclei, central gray, substantia nigra pars compacta, locus coeruleus, pontine reticular nucleus and cerebellum. Distribution pattern was remarkably different from that of [125I]VIP binding sites in the hippocampal formation, lateral hypothalamic area, substantia nigra pars compacta, pontine reticular nucleus and cerebellum. The present results suggest that PACAP may have a physiological role in the regulation of the central nervous system.


Morphological and electrophysiological evidence for postsynaptic localization of functional oxytocin receptors in the rat dorsal motor nucleus of the vagus nerve. The vagal complex is innervated by oxytocin immunoreactive axons of hypothalamic origin. The presence of oxytocin binding sites in the dorsal motor nucleus of the vagus nerve of the rat was evidenced by autoradiography with a radioiodinated oxytocin antagonist as ligand. Two weeks following a unilateral vagotomy, distal to the nodose ganglion, binding sites were reduced below the level of detection in the ipsilateral dorsal motor nucleus of the vagus nerve. Choline acetyltransferase immunoreactivity was also markedly reduced in the vagal motoneurons whose axons had been transected. Electrophysiological studies were performed in vitro in brainstem slices from control rats. In antidromically identified vagal motoneurones, oxytocin applied at 0.1-1.0 microM either caused a reversible depolarization or generated, under voltage-clamp conditions, a transient inward current. These responses persisted under the condition of synaptic uncoupling. Taken together these observations favour the notion that oxytocin of hypothalamic origin acts directly on rat vagal motoneurones.


After chronic opioid exposure sensory neurons become supersensitive to the excitatory effects of opioid agonists and antagonists as occurs after acute elevation of GM1 ganglioside. Mouse sensory dorsal-root ganglion (DRG) neurons chronically exposed to 1 microM D-Ala2-D-Leu5-enkephalin (DADLE) for greater than 1 week in culture become tolerant to opioid inhibitory effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD). Acute application of higher concentrations of DADLE (ca. 10 microM) to these treated neurons not only fails to shorten the APD but, instead, generally elicits excitatory effects, i.e. prolongation of the APD. The present study shows that chronic DADLE- or morphine-treated DRG neurons also become supersensitive to the excitatory effects of opioids. Whereas nM concentrations of dynorphin(1-13) are generally required to prolong the APD of naive DRG neurons, fM levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine do not alter the APD of naive DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the treated cells. Similar supersensitivity to the excitatory effects of opioid agonists and antagonists was previously observed after acute treatment of naive DRG neurons with GM1 ganglioside. Our results suggest that both chronic opioid and acute GM1 treatments of DRG neurons greatly enhance the efficacy of opioid excitatory receptor functions so that even the extremely weak agonist properties of naloxone and diprenorphine become effective in prolonging the APD of these treated cells when tested at low concentrations, whereas their antagonist properties at inhibitory opioid receptors do not appear to be altered. Furthermore, whereas cholera toxin-B subunit (CTX-B; 1-10 nM) blocks opioid-induced APD prolongation in naive DRG neurons (presumably by interfering with endogenous GM1 modulation of excitatory opioid receptors functions), even much higher concentrations of CTX-B were ineffective in chronic opioid-treated as well as acute GM1-elevated neurons. These and related data suggest that opioid excitatory supersensitivity in chronic opioid-treated DRG neurons may be due to a cyclic AMP-dependent increase in GM1 ganglioside levels. Our results may clarify mechanisms of opioid dependence and the paradoxical supersensitivity to naloxone which triggers withdrawal symptoms after opiate addiction.


Area postrema mediation of physiological and behavioral effects of lithium chloride in the rat. The area postrema (AP), a chemoreceptor trigger zone for nausea and vomiting, has been implicated in taste aversion conditioning with LiCl. In addition to taste aversion acquisition, the present studies indicate that a number of other responses to LiCl administration are eliminated by lesions of the AP. These include a behavioral response, 'lying-on-belly' as well as two physiological responses, delayed stomach emptying and hypothermia. These findings suggest that the area postrema is critically involved in the detection of LiCl and in a wide range of responses to this toxin. They also provide strong evidence that the failure to acquire conditioned taste aversions to LiCl-paired flavors after AP lesions can be attributed to the absence of a significant 'illness' response in lesioned animals.


Pinealectomy and constant illumination increase the density of melatonin binding sites in the pars tuberalis of rodents. We report here the effects of pinealectomy and light exposure on the melatonin receptor density in the pars tuberalis of the rat and the European hamster using quantitative autoradiography. Scatchard analysis revealed that 24 and 72 h of constant light exposure (LL) before sacrifice did not modify the Kd value of melatonin for its receptors in rats and European hamsters (about 70 pM). In contrast, the Bmax value was significantly increased in both species when the animals were kept in constant illumination for 72 h before sacrifice (50%-70% compared with the controls). A similar increase was also observed in rats pinealectomized 3 days before sacrifice and then kept in either constant illumination or in 12L/12D. Pinealectomy or constant light exposure are known to result in a clear decrease in the concentration of circulating melatonin. We demonstrate here that they also result in an increase in the density of melatonin receptors. This could suggest a direct effect of melatonin on its own receptors.


Effects of diabetes on stress-induced analgesia in mice. The present studies were designed to determine whether streptozotocin-induced (STZ-induced) diabetes in mice can attenuate the development of antinociception induced by exposure to both foot shock and forced swimming stress. Foot shock stress produced significant analgesia both in control and diabetic mice. However, the extent of foot shock stress-induced analgesia (FSSIA) in diabetic mice was significantly lower than that in control mice. Naloxone (2 mg/kg, i.p.) significantly attenuated FSSIA in control mice, but was without effect on FSSIA in diabetic mice. One-minute swimming stress had no significant effect on tail-pinch latency in control mice, whereas 3-min swimming stress produced significant analgesia in these mice. Diabetic mice exhibited robust swimming stress-induced analgesia (SSIA): one-min swimming stress produced significant analgesia in diabetic mice. These analgesic effects were blocked by naltrindole, a selective antagonist of delta-opioid receptors, but not by pretreatment with beta-funaltrexamine, an irreversible and selective antagonist of mu-opioid receptors. These results suggest that the deficiency in the functioning of mu-opioid receptors caused by diabetes results in significant activation of an endogenous analgesic system, which is mediated mainly by delta-opioid receptors.


[3H]nipecotic acid binding to GABA uptake sites in human brain. The binding of [3H]nipecotic acid to frozen post-mortem human brain tissue has been characterized. Competition experiments with gamma-aminobutyric acid (GABA), GABA uptake inhibitors, ligands active at post-synaptic GABA receptors and receptors for other neurotransmitter systems, suggest that [3H]nipecotic acid binds to the neuronal (but not glial) GABA uptake site. Competition and kinetic experiments suggest that 85% of the binding is to a high affinity site. The dissociation constants (Kd) measured in kinetic and equilibrium experiments were in the same range (0.5-0.6 microM). The regional distribution was studied in 19 brain regions and the binding was relatively homogenous. It is concluded that [3H]nipecotic acid binding can be used as a marker for neuronal GABA uptake sites in post-mortem human brain tissue.


The presence of thromboxane A2 receptors in cultured astrocytes from rabbit brain. We have previously shown that human astrocytoma cells (1321N1) express thromboxane A2 (TXA2) receptors, of which stimulation activates phosphoinositide hydrolysis (Nakahata et al., Eur. J. Pharmacol. 162 (1989) 407). In order to examine whether TXA2 receptors exist in native astrocytes or not, rabbit cultured astrocytes were used. Glial fibrillary acidic protein (GFAP)-positive astrocytes were obtained three weeks after culture of brain. [3H]ONO NT-126, a TXA2 antagonist, bound to the membranes derived from cultured rabbit astrocytes with the dissociation constant (Kd) of 0.23 nM and the maximum binding site (Bmax) of 69.5 fmol/mg protein. STA2, a stable TXA2 receptor agonist, activates phosphoinositide hydrolysis in a concentration-dependent manner, and S-145, a TXA2 antagonist, inhibited STA2-induced phosphoinositide hydrolysis. The results indicate that TXA2 receptors exist in cultured rabbit astrocytes and the activation of TXA2 receptors results in phosphoinositide hydrolysis.


Ontogenic study of lithium-pilocarpine-induced status epilepticus in rats. Lithium is known to potentiate the ability of pilocarpine to induce status epilepticus in rats. The goal of this study was to determine whether lithium could potentiate pilocarpine-induced seizures in developing animals. Behavioral, electroencephalographic (EEG), and histopathological changes induced by systemic administration of lithium (3 meq/kg) followed 20 h later by pilocarpine (3, 10, 30, 60 mg/kg) were studied in 3-30-day-old rats. Lithium followed by pilocarpine (30 and 60 mg/kg) induced hyperactivity, tremor, loss of postural control and scratching but no electrographic seizures in 3-8-day-old rats. In the 7-10-day-old animals pretreatment with lithium and pilocarpine 60 mg/kg induced status epilepticus with sustained myoclonus and continuous bilateral synchronous spike and sharp wave, but doses of pilocarpine lower than 60 mg/kg had no effect. The susceptibility to lithium-pilocarpine-induced status epilepticus increased markedly during the third postnatal week of life. During this time period, rats treated with lithium (3 meq/kg) plus pilocarpine 10 mg/kg exhibited behavioral and EEG manifestations of status epilepticus. The same combination of lithium and pilocarpine failed to induce status epilepticus either before or after the third week of life. Histopathological analysis of the brains of the animals used in these studies failed to demonstrate the widespread damage reported in adult rats that have undergone lithium-pilocarpine-induced status epilepticus.


5-Hydroxytryptamine receptor agonists for the abortive treatment of vascular headaches block mast cell, endothelial and platelet activation within the rat dura mater after trigeminal stimulation. Antidromic stimulation of small caliber trigeminal axons causes neurogenic inflammation in the dura mater and tongue as evidenced by marked increases in mast cell activation, protein extravasation, as well as in the numbers of endothelial cytoplasmic vesicles, endothelial microvilli and platelet aggregates within ipsilateral post-capillary venules. In this report, we examined the effects of pretreatment with serotonin1 receptor agonists, dihydroergotamine (50 micrograms/kg, i.v.) and sumatriptan (100 micrograms/kg, i.v.) on the light and electron microscopic changes which develop after trigeminal ganglion stimulation. Both dihydroergotamine and sumatriptan are useful in the acute treatment of vascular headaches and bind with high affinity to 5-HT1D receptors. Both drugs decreased significantly the number of dural vessels showing endothelial or platelet changes and the numbers of activated mast cells, but did not affect the neurogenic response in the tongue. The drugs also blocked the accumulation of horseradish peroxidase reaction product within the endothelium and perivascular space on the stimulated side. The receptor is not present on trigeminovascular fibers innervating extracranial cephalic tissues. Drug mechanism probably involves inhibition of a proximal step in the pathophysiological cascade (e.g., via activation of a prejunctional receptor) because (a) receptors for sumatriptan have not been identified on mast cells whereas the inflammatory response was attenuated in mast cells as well as within platelets and the endothelium and (b) previous work indicates that sumatriptan and dihydroergotamine block neurotransmitter release. Hence, constriction of vascular smooth muscle mediated by postjunctional 5-hydroxytryptamine receptors is unlikely to explain the anti-inflammatory actions of dihydroergotamine or sumatriptan reported here.


Beta-adrenergic receptor activity of cerebral microvessels in experimental diabetes mellitus. The effect of diabetes mellitus on beta-adrenergic receptor number (B(max)), receptor-cyclase coupling and adenylate cyclase (AC) activity was determined in cerebral microvessels isolated from control and streptozotocin induced diabetic rats after 5 weeks of induction of diabetes. Scatchard analysis of [125I]iodocyanopindolol (ICYP) binding indicated that the B(max) (fmol/mg) in diabetic rat cerebral microvessels (63.8 +/- 4.8) (mean +/- S.E.M.) was not significantly different from the B(max) in control rats (56.5 +/- 6.9). Isoproterenol competition of [125I]ICYP binding sites indicated that the percentage of beta-receptors expressing high affinity binding was 53.9 +/- 0.45% in control rats and 47.5 +/- 2.3% in diabetic rats. The total isoproterenol-stimulated AC activity (pmol cAMP/mg) in diabetic rats (76.7 +/- 6.1) was significantly lower than that in control rats (118.4 +/- 11.2) (P less than 0.01). However, the net isoproterenol-stimulated AC activity (i.e. total minus GTP-stimulated AC activity) was not altered in diabetes. The net sodium fluoride (NaF) stimulated AC activity in diabetic rats (109.5 +/- 11.4) was significantly lower than the control rats (154.3 +/- 16.3) (P less than 0.05). It is concluded that diabetes mellitus in rats is associated with reduced post receptor activation of adenylate cyclase in cerebral microvessels while the beta-adrenergic receptor density, affinity and receptor-cyclase coupling are not significantly altered.


Social isolation increases the density of [125I]omega-conotoxin GVIA binding sites in the rat frontal cortex and caudate nucleus. Rats were reared from weaning either in isolation or in social groups for 12 weeks. Potential isolation-related changes in L- and N-type voltage-sensitive calcium channels (VSCCs) were assessed by the in vitro binding of [3H]isradipine (100 pM) and [125I]omega-conotoxin GVIA (4 pM) to membranes prepared from three discrete central nervous system regions: frontal cortex, caudate nucleus and hippocampus. The [3H]isradipine binding was generally not affected by isolation. However, [125I]omega-conotoxin GVIA binding was significantly higher in frontal cortex (52%) and caudate nucleus (75%) of isolated rats when compared with socially reared controls. The increased [125I]omega-conotoxin GVIA binding reflected an elevated density of binding sites without an alteration of receptor affinity. The possible contribution of an increased density of neuronal N-VSCCs (as labeled with [125I]omega-conotoxin GVIA) to the behavioral and neurochemical changes observed in 'isolation syndrome' is discussed.


Differentiation of Neuro-2a neuroblastoma cells by an antibody to GM3 ganglioside. A monoclonal antibody against GM3 ganglioside (GM3Ab) was found to trigger differentiation of Neuro-2a cells in culture. The differentiation of Neuro-2a cells by GM3Ab was accompanied by increased levels of intracellular serotonin and amino acid neurotransmitters viz. aspartate, glutamate, glutamine, glycine and taurine. Further study indicated that the increase in the serotonin level was not due to a higher rate of serotonin synthesis but rather to a higher rate of active transport of serotonin from the medium. Studies on the cell surface gangliosides revealed that unlike the proliferating cells, the GM3Ab-mediated differentiated cells contained higher gangliosides in addition to GM3 and GM2 gangliosides. Analysis of total cellular proteins indicated the appearance of a 25 kDa protein, pI 5.4, in the GM3Ab-treated cells--a small amount of this protein was observed in dibutyryl cAMP (Bt2cAMP)-treated cells, however, the protein was totally absent in the 5-bromo-2'-deoxyuridine (BrdU)-treated cells. Investigation of the mode of action of GM3Ab indicated that the cellular differentiation was due to increased cAMP accumulation resulting from an increase in the adenylate cyclase activity. Further studies with different agents affecting protein kinase C (PKC) activity and direct assay of PKC ruled out the possibility that GM3Ab mediated its effect via PKC. This GM3Ab-induced differentiation could be inhibited by protein kinase A (PKA) inhibitor, H8, but could not be inhibited by sphingosine, an inhibitor of PKC. Pertussis toxin could mimic the effect of GM3Ab, suggesting that GM3Ab caused the elevation in the adenylate cyclase activity by reducing the Gi-protein inhibition of the adenylate cyclase. The data suggests that GM3Ab, after interaction with cell surface GM3, elevated intracellular cAMP level by withdrawing the inhibitory effect of some undefined factor(s) present in culture medium which normally keeps adenylate cyclase activity low through activation of Gi-protein.


Current hypotheses of structural pattern formation in the somatosensory system and their potential relevance to humans. Current hypotheses of structural pattern formation in the mammalian somatosensory system are modeled on experimental findings from the trigeminal system of rodents. The present results show that, like rodents, the trigeminal nucleus principalis of humans contains a parcellated pattern of cytochrome oxidase dense patches. These results provide an indication of the potential usefulness of rodent-based hypotheses for understanding pattern formation in human somatosensory connections.


Modification of voltage-dependent Na+ current by triphenyltin, an environmental pollutant, in isolated mammalian brain neurons. In order to reveal the profile of neurotoxicity of triphenyltin on the mammalian central nervous system (CNS) through the modification of a voltage-dependent Na+ channel, the effects of triphenyltin on the kinetics of voltage-dependent Na+ current (INa) were examined in acutely dissociated pyramidal neurons of rat hippocampus. Triphenyltin at the concentration of 1.10(-6) M decreased both the time to peak and the half-decay time of the INa without affecting the current-voltage relationship. Triphenyltin moved the steady-state inactivation curve to a depolarizing direction. In the presence of triphenyltin, the reactivation of Na+ channel inactivated during the depolarization occurred quicker than that of the control at the pulse intervals of less than 10 ms. It can be suggested that triphenyltin potentially increases the cell excitability in mammalian CNS through the modification of the INa.


Strain comparisons and developmental profile of the delta subunit of the murine GABAA receptor. GABAA receptors are multisubunit inhibitory chloride channels in the brain which open in response to binding of gamma-aminobutyric acid (GABA) and are thought to be involved in some forms of seizures. We compare the sequence and expression of the GABAA receptor delta subunit in audiogenic seizure prone (DBA/2J) and seizure resistant (C57BL/6J) inbred strains of mice and also report this subunit's postnatal developmental profile. We did not detect any unique features in the delta subunits of DBA/2J mice which might explain their seizure susceptibility, but did detect in some clones from both DBA/2J mice and C57BL/6J mice an unusual substitution of His for a conserved Tyr in the delta subunit's first putative transmembrane region.


Positive feedback action of pituitary beta-endorphin on acupuncture analgesia afferent pathway. Potentials in the final sector of the afferent pathway from the acupuncture point (AP) were enhanced by intraperitoneal 0.5 mg/kg morphine without changing the threshold of AP stimulation and greatly decreased by hypophysectomy. The decreased potentials were restored to the control level by morphine (0.5 mg/kg, IP). Potentials evoked in the final sector of the afferent pathway from the nonacupuncture point (NAP) by NAP stimulation after lesion of the analgesia inhibitory system were greatly enhanced by corticotropin (ACTH) (0.25 mg/kg, IP) and greatly decreased by hypophysectomy. Diminished potentials were restored to the control level by ACTH (0.25 mg/kg, IP). Both morphine (0.5 mg/kg, IP) and ACTH (0.25 mg/kg, IP) produced analgesia, but morphine did not affect acupuncture analgesia (AA) and ACTH did not affect nonacupuncture point stimulation-produced analgesia (NAA). All analgesia, that due to 0.5 mg/kg morphine or 0.25 mg/kg ACTH, AA, and NAA were abolished by hypophysectomy. The abolished AA and NAA were restored by 0.5 mg/kg morphine and 0.25 mg/kg ACTH, respectively. Hence, beta-E and ACTH liberated from the pituitary gland by stimulation of an AP and NAP may act as positive feedback on the AA and NAA afferent pathways, respectively.


Cocaine-induced ACTH secretion: dependence of plasma levels of the drug and mode of exposure. To determine whether changes in pituitary responsiveness might account for the lack of corticotropin (ACTH) stimulation following 6 consecutive days of continuous cocaine administration (5 or 25 mg/kg/day, via osmotic minipumps), the hormonal response of vehicle- or cocaine-pretreated male rats was compared. Intravenous injections of synthetic corticotropin-releasing factor (CRF) (0.2, 1, or 5 micrograms/kg) elicited dose-dependent increases in ACTH secretion irrespective of whether rats had been previously exposed to cocaine or not. Similarly, in both vehicle- and cocaine-pretreated rats ACTH response to acute injections of the drug was identical, indicating that pituitary corticotrophs remained responsive following continuous administration of cocaine. To determine and compare plasma concentrations of cocaine and its metabolites after continuous or acute administration of the drug, pharmacokinetics analysis of concentration vs. time was ascertained. Circulating concentrations of cocaine from rats continuously exposed to the drug were relatively low throughout the 6 days of exposure. In contrast, intravenous injections of cocaine produced peak concentrations of the drug that were significantly higher than those measured during continuous cocaine infusion. Such peak concentrations in cocaine correlated with marked increases in plasma ACTH levels. Plasma concentrations of the metabolites benzoylecgonine and methyl ester ecgonine followed a pharmacokinetic clearance similar to that of the parent compound, with low concentrations detected during continuous exposure whereas high concentrations were observed following intravenous injections of the drug. Our results suggest two nonmutually exclusive conclusions. First, there may be a critical threshold of cocaine plasma concentrations (as indicated by our results as being over 800 ng/ml) that are necessary for activation of the hypothalamic-pituitary-adrenal axis to occur.(ABSTRACT TRUNCATED AT 250 WORDS)


Biochemical and behavioral effects of steroids on GABAA receptor function in long- and short-sleep mice. The in vitro and in vivo effects of alphaxalone, a steroid anesthetic, and two physiological steroids, tetrahydrodeoxycorticosterone (THDOC) and pregnenolone sulfate (PS), on GABAA receptor function were evaluated in long-sleep (LS) and short-sleep (SS) mice. In vitro, both alphaxalone and THDOC enhanced GABAergic inhibition as measured by [3H]FNZ binding and GABA-stimulated 36Cl- flux. However, with the exception of alphaxalone potentiation of [3H]FNZ binding, which was greater in SS brain regions, LS and SS mice did not differ in their degree of enhancement. Pregnenolone sulfate produced mixed agonistic and antagonistic effects on GABAergic function, dependent upon brain region, with few differences between the lines of mice. In vivo effects of these steroids on sleep time indicated that, like other anesthetic agents, THDOC and alphaxalone induced longer sleep times in LS mice. Antagonism by PS of ethanol-induced sleep time was observed in LS mice only; however, this effect was dependent upon the dose of ethanol used and on the vehicle used to prepare the steroid. Pentobarbital-induced sleep time was not reduced by PS treatment in either line of mouse. These results demonstrate that few differences in sensitivity of the GABAergic receptor to these steroids exist between LS and SS mice. Thus, unlike the differences between LS and SS mice in GABAergic mediation of responses to ethanol and benzodiazepines, there is little genetic variability in subtypes of GABAA receptors capable of modulation by steroids in these lines of mice.


An analysis of male and female Olympic swimmers in the 200-meter events. Videotapes of the swimming events at the 1988 Seoul Olympics were analyzed to obtain the stroke rate (SR) and stroke length (SL) for each swimmer. In this paper, SR and SL data from the 200-m events were combined with anthropometric data (height and age of the swimmers) and the final time achieved by each swimmer. Statistical analyses were then carried out to determine the relationships among the different variables and their correlation with final times. Differences in performances between male and female swimmers were also investigated. Stroke lengths were found to have high correlations with the final times achieved by the competitors. A strong relationship was also found between height and final time. On an average, the men were found to have a lower stroke rate and to be faster than the women. The latter finding could be a result of the men's longer stroke length and greater height.


Metabolic changes during serial squash matches in older men. We have previously reported dramatic changes in heart rate and blood biochemistry in older men during and shortly after competitive squash. In this study we sought to determine whether these changes are attenuated or exaggerated during tournament matches played in rapid succession. Ten veteran (greater than 45 yrs) players were studied during three competitive matches played over a 36-hr period. Squash was associated with significant changes in heart rate and circulating concentrations of catecholamines, lactate, free fatty acids, and potassium. These changes were of equal magnitude and in some cases tended to be exaggerated during the second and third matches. These data confirm the acute changes in cardiac function and metabolism that occur during competitive squash and suggest that these responses are not down-regulated but may in fact be accentuated during sequential tournament matches.


Selection of a maximal test protocol to validate the Canadian Aerobic Fitness Test. The purpose of this study was to select a maximal aerobic power test protocol with which to validate a submaximal step test prediction equation. Subjects (N = 129), males and females 15 to 69 years of age, performed one maximal step test and one maximal treadmill test. The maximal treadmill protocol yielded higher peak VO2, ventilation, heart rate, and RER values. An age-predicted maximum heart rate was achieved by 35% of subjects on the step test and 55% on the treadmill. An RER of 1.15 was attained by 22% of the subjects on the step test and 53% on the treadmill. Regression analysis indicated that the submaximal test was more predictive of VO2max when the maximum was attained using the treadmill rather than the maximal step protocol. Peak VO2 values obtained from subjects tested on the treadmill will be used to develop a new prediction equation for the Canadian Aerobic Fitness Test.


Relationship among selected performance measures on the Omnitron Total Power Unit. The present study assessed the interrelationships among selected performance parameters on the Omnitron Total Power Unit. Forty-eight males (18 to 31 yrs) repeated on 2 days the chest press, shoulder press, lat row, and lat pull-down exercises at low, medium, and high levels of resistance. Test-retest reliability for peak force, average linear velocity, and average power output ranged form r = 0.71 to r = 0.97 (p less than 0.05) (29 of 36 correlations greater than or equal to 0.9), and demonstrate that the different testing protocols provide reliable individual differences for assessing muscular performance. The average linear velocity and average power output data provide little new information about muscular performance besides that attained from peak force analysis. The percentage common variance among the levels of resistance and output modes ranged from 90 to 98%. Thus, assessing peak force at a single level of resistance on the Omnitron is sufficient to obtain an overall assessment of muscular performance.


Exercise-induced catabolic responses in various muscle fibres. In Wistar rats a 10-hr swim caused an immediate increase of 3-methylhistidine and free tyrosine content in the white portion of quadriceps muscle. In the red portion the elevated levels of these amino acids were observed within 6 to 48 hrs of postexercise recovery. The glycogen depletion was substantially higher in the red portion of the muscle, indicating that the most active were oxidative-glycolytic fibres during the exercise. In conclusion, during exercise the most active muscle fibres do not contribute to the mobilization of protein resources. In a later part of postexercise recovery, catabolic changes occur in most active fibres, constituting, reasonably, a part of enhanced protein turnover.


The effect of interval and continuous training on the aerobic parameters. The purpose of this study was to investigate changes in the parameters of aerobic function resulting from continuous training (CT) and interval training of both low power (LPO-IT) and high power output (HPO-IT). Untrained males (n = 17, 25.1 yrs) trained 10 weeks on cycle ergometers (four 40-min sessions a week) at 80% VO2max. Cycle ramp function tests at 0 and 10 weeks were used to determine the four aerobic parameters:VO2max, ventilation threshold (VeT), effective time constant for O2 uptake kinetics (MRT), and work efficiency (eta): VO2max increased significantly (3.30 to 3.66 l.min-1). Absolute VeT increased significantly (2.17 to 2.45 l.min-1) but there was no change in the relative threshold (VeT/VO2max). MRT decreased significantly from 38.8 to 33.1 seconds and there was no change in eta. There were no between-group differences; thus neither low power output nor high power output interval training offers an advantage over continuous training of the same average power output in altering the aerobic parameters.


Cognitive strategies of university athletes. Thirty-six college basketball players and skiers, both men and women, were interviewed about their use of cognitive processes to prepare themselves for competition. The interviews examined cognitive techniques for enhancing motor skills and developing competitive strategies. During the interviews the subjects engaged in some of their cognitive preparation strategies and then described the details of these experiences. All athletes reported that the use of cognitive strategies enhanced their performance. Their imagery was rich, detailed, and multisensory. An average of three sensory modalities were present in the imagery. Vision, kinesthesis, and touch were the most common sensory experiences reported, but audition, taste, and smell were also experienced by some athletes. Strong affective states, especially confidence and satisfaction, accompanied the imagery. Individual differences in the imagery used by the athletes were also found and may be related to individual differences in the athletes' cognitive styles.


Fatigue and changes of ATP, creatine phosphate, and lactate during the 400-m sprint. Fatigue during the 400-m sprint was studied by measuring muscle ATP, creatine phosphate (CP), lactate (M-La), and blood lactate (B-La) in six male runners before and after four experimental sprints (100, 200, 300, and 400 m). During the first 100 m, muscle CP decreased from 15.8 +/- 1.7 to 8.3 +/- 0.3 mmol/kg while M-La increased to 3.6 +/- 0.4 mmol/kg. After 200 m the CP had decreased to 6.5 +/- 0.5 mmol/kg and M-La had increased to 8.3 +/- 1.1 mmol/kg. At the end of the 400 meters, ATP and CP concentrations had decreased by 27% and 89%, respectively, and M-La had increased to 17.3 +/- 0.9 mmol/kg. It was concluded that after 200 m the speed of running decreased, although CP was not depleted and lactate concentration was not at maximum level. Complete fatigue occurred when CP stores were depleted and B-La and M-La attained an individual maximum.


[The effects of positive, negative and neutral self-talk on motor performance]. The purpose of this study was to examine the influence of self-talk on motor performance. Forty-six (46) male and female college students from the Ivory Coast were randomly assigned to two experimental groups (N = 15 for each) and one control group (N = 16). All subjects completed 5 blocs of 10 trials of a dart throw on a 45-cm by 45-cm target. The distance from the center of the target was recorded by measuring the distance along the x and y axes. Subjects in one experimental group (VP) were asked to repeat aloud positive verbalizations in between blocs of trials, while subjects in the other experimental group (VN) repeated aloud negative verbalizations in between blocs of trials. The control group (T) rested between trials. Results indicated that the angular errors in the VP group decreased faster than those of the T group, that the performance of the VN group did not improve significantly, and that the rate of angular error in the VN group was higher than that of the T and VP groups.


Blood lactate response to the Canadian Aerobic Fitness Test in females. This study evaluated the blood lactate concentration ([LA-]) response to the Canadian Aerobic Fitness Test (CAFT) in female subjects and compared the strength of prediction of VO2max determined by [LA-] and heart rate (HR). The sample was composed of 98 Canadian Forces females between the ages of 18 and 45 years. The [LA-] after each stage of the step-test was measured in all subjects by sampling blood from the fingertip. VO2max was measured directly during a maximal treadmill run in 66 of these subjects. The results showed that increasing stages of the step-test were associated with increasing [LA-]. The correlation between [LA-] after Stage 5 of the step-test and the directly determined VO2max was r = -0.72 and did not differ significantly from the correlation between HR and VO2max (r = -0.66). The relationship between [LA-] and VO2max for these females was similar to the one established earlier for males; however, the correlation between HR and VO2max for females was different from that observed in males. The present data for the females suggest that [LA-] and HR after Stage 5 of the CAFT predict VO2max equally well for females under age 40.


Fat metabolism, exercise, and the cold.

Fat metabolism, exercise, and the cold. Whereas short-term cold exposure depletes glycogen reserves, repeated and prolonged moderate exercise in a cold environment creates an energy deficit that is satisfied by an increased metabolism of depot fat. Factors contributing to the fat loss include an exercise-induced hypertrophy of lean tissue, a loss of energy through a cold-induced ketonuria, a stimulation of resting metabolism, increases in the energy cost of movement, and a lower yield of energy per litre of oxygen consumed. Biochemical explanations of the enhanced lipolysis include increased catecholamine secretion, altered sensitivity of catecholamine receptors, and decreases of circulating insulin. The enhanced fat loss with combinations of cold and exercise may be helpful in the therapy of obesity, although the response seems less well developed in women than in men. Moreover, there may be other objections to cold exposure in an older obese population. Short-term glycogen depletion has negative implications for the endurance competitor. Cold acclimation, by favoring an insulative response to cold, reduces glycogen depletion; endurance training may supplement this effect by enhancing the activity of fat-metabolizing enzymes.


Design of a calf muscle ergometer for the study of local muscle blood flow. A simple ergometer is described that allows highly reproducible plethysmographic measurements of blood flow to be made within 10 seconds of ceasing rhythmic quantitated exercise of the calf muscle. A power output of some 25 Watts is developed with a mechanical efficiency of some 20%. The device seems applicable to a variety of clinical assessments.


Evaluating the influence of sleep deprivation upon circadian rhythms of exercise metabolism. Cardiorespiratory and gas exchange responses to a moderate, standardized treadmill walking task showed a weak circadian rhythm, with larger superimposed peaks attributable to feeding. However, both rhythms became progressively attenuated during a period of sleep deprivation. A method of exploring this phenomenon is illustrated by an analysis of data on walking heart rate, respiratory minute volume, oxygen intake, and rating of perceived exertion, collected on 11 young men at 3-hr intervals during 60 hours of sleep deprivation.


[Attentional requirements for preparing to return a serve in tennis]. Recent studies demonstrated that athletes use more efficient strategies than novices in sports with high perceptual requirements (Abernethy and Russel, 1984; Goulet et al., 1989; Starkes, 1987b). The aim of the present study was to investigate the attentional cost of information processing preceding action in tennis players of different calibers. The dual-task paradigm was used. The primary task consisted of identifying the type of serve (flat, slice, or top-spin) presented on 16-mm file. The secondary task was a manual response to an auditory probe. Results demonstrated that attentional requirements during information processing do not differ between experts and novices. Nevertheless, the experts' results on the primary task are significantly higher than those of novices, whether the primary task is performed alone or simultaneously with the secondary task. The attentional cost of information processed during the ritual phase of the serve is significantly higher than the costs for processing information from the preparation and execution of the serve. It appears, therefore, that attentional input effort is maximal before identification of the most important cues necessary for adequate performance.


[Effects of oxygen free radicals on the function of the cardiac sarcoplasmic reticulum]. Peroxidative stress, exerted by oxygen free radicals, seems to be an important mechanism of the ischemia-reperfusion myocardial damage. In the present study we evaluated the modifications of sarcoplasmic reticulum function subjected to peroxidation by ferric ions. A subcellular fraction enriched in sarcoplasmic reticulum was obtained from rabbit hearts by homogenization and differential centrifugations. Sarcoplasmic reticulum vesicles were peroxidated through incubation for 5 min at 37 degrees C in presence of ferric cloride (FeCl3) ranging in concentration between 0.3 and 0.9 mM. Peroxidation of sarcoplasmic reticulum vesicles determined a dose-dependent reduction of Ca-uptake (39.2 +/- 10.3, 36.5 +/- 9.9, 28.9 +/- 8.4 and 18.8 +/- 8.2 nmol/min/mg in presence of 0, 0.3, 0.6 e 0.9 mM FeCl3; NS, p less than 0.05 and less than 0.01, respectively) which was paralleled by an increase in the production of malondialdehyde, an index of lipid peroxidation (1.0 +/- 1.0, 7.0 +/- 3.2, 14.1 +/- 3.9 and 27.0 +/- 4.7 nmol/mg in presence of 0, 0.3, 0.6 e 0.9 mM FeCl3; p less than 0.05, less than 0.01 and less than 0.01, respectively). Depression of Ca-uptake was not accounted for by modifications of Ca-ATPase activity or membrane aspecific permeability to Ca++ ions, since these parameters were not affected by exposure to 0.3-0.9 mM FeCl3. On the contrary, the responsiveness of Ca-release channels to the specific inhibitor ryanodine was greatly altered, even at lower FeCl3 concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)


In situ hybridization to the Crithidia fasciculata kinetoplast reveals two antipodal sites involved in kinetoplast DNA replication. Kinetoplast DNA is a network of interlocked minicircles and maxicircles. In situ hybridization, using probes detected by digital fluorescence microscopy, has clarified the in vivo structure and replication mechanism of the network. The probe recognizes only nicked minicircles. Hybridization reveals prereplication kinetoplasts (with closed minicircles), donut-shaped replicating kinetoplasts (with nicked minicircles on the periphery and closed minicircles in the center), and postreplication kinetoplasts (with nicked minicircles). Replicating kinetoplasts are associated with two peripheral structures containing free minicircle replication intermediates and DNA polymerase. Replication may involve release of closed minicircles from the center of the kinetoplast and their migration to the peripheral structures, replication of the free minicircles therein, and then peripheral reattachment of the progeny minicircles to the kinetoplast.


[Serological verification of the role of enteroviruses in the etiology of polyradiculoneuritis and aseptic meningoencephalitis]. In a group of 38 patients with the Guillain-Barré syndrome (SGB) and a group of 38 patients selected at random who suffered from meningoencephalitis the authors compared the characteristics by age, sex, onset of the disease (month of the year) and intervals between the onset of the disease and collection of serum samples. Using the neutralization microtest, the sera were examined for antibodies against 23 serotypes of enteroviruses incl. polioviruses. The groups differed significantly in the following indicators: the SGP group had a non-seasonal annual distribution, it included different age groups, there were longer intervals of sample collection. There were fewer cases with an assessed enteroviral aetiology, no case with a polioviral aetiology. More detailed analysis of serological results revealed, that the group does not differ fundamentally as regards the immunological experience of individuals with enteroviruses before the disease. The authors discuss the need of more detailed examinations of the enteroviral aetiology in all cases of SGB.


Formation of superoxide in the reaction of photolytically altered nifedipine--a nitroso compound--with unsaturated membrane lipids. Nifedipine, which is unstable at light, is photolytically converted to the corresponding 4-[2'-nitrosophenyl]-pyridine (NTP). We reported earlier that NTP react with unsaturated lipids in a pseudo Diels-Alder reaction, thus forming stable nitroxide radicals. In this paper we report that superoxide is being generated in the latter reaction. Superoxide formation was evidenced by SOD-inhibitable cytochrome c reduction in the reaction of NTP with egg phosphatidylcholine at molar ratio 1:1, and 1:3. In this reaction an ESR-observable nitroxide radical was formed. Maximum nitroxide formation was observed after 90 min; the addition of SOD (93 units/ml) increased the concentration of nitroxide. This effect of SOD was reversed by catalase, indicating involvement of hydrogen peroxide in this effect. The nitroxide radical formation appears to be metal-independent, since neither iron salts, nor an iron chelator, desferal, influenced the nitroxide formation. Although production of superoxide in our system was only observed at high concentrations of NTP and of unsaturated lipids, this reaction may be of potential cytotoxic significance due to redox cycling of the nitroxide/hydroxylamine couple in cellular systems.


Decoding calcium signals by multifunctional CaM kinase. Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is one of the three major protein kinases coordinating cellular responses to hormones and neurotransmitters. It mediates the action of Ca2+ on neurotransmitter synthesis and release, on carbohydrate metabolism and on the cytoskeleton. CaM kinase has structural/functional properties that facilitate its response to distinctive attributes of Ca2+ signals which often involve transient increases that span a narrow concentration range and increases that are pulsatile rather than persistent. The kinase responds to the narrow working range of Ca2+ signals by the use of calmodulin as the Ca2+ sensor. It is activated by the binding of calmodulin to an autoinhibitory domain that keeps the kinase inactive in the basal state. The transient nature of the signal is accommodated by autophosphorylation of this autoinhibitory domain which allows the kinase to remain partially active after calmodulin dissociates and thereby switches it to a Ca(2+)-independent species. The pulsatile nature of Ca2+ signals may also be decoded by CaM kinase. Autophosphorylation traps calmodulin on autophosphorylated subunits by greatly reducing its off-rate. At high frequency of stimulation, calmodulin would remain trapped during the brief interval between Ca2+ oscillations and each successive rise in Ca2+ would recruit more calmodulin. This may enable a stimulus frequency dependent activation of CaM kinase.


DNA ploidy of primary hepatocellular carcinoma and pulmonary metastases. To better comprehend the differences in deoxyribonucleic acid (DNA) content between a primary hepatocellular carcinoma (HCC) and pulmonary metastatic nodules, tissue specimens taken at autopsy of 25 patients who had not received any drugs to treat the malignancy were examined using microspectrophotometry. The DNA distribution patterns were classified into Types I-III, and low (Types I and II) or high (Type III) ploidies, according to DNA distribution. Changes in the DNA content from high to low ploidies, namely DNA ploidy reduction from the primary lesion to pulmonary metastatic lesions, was evident in 9 of the 25 patients (36%), and changes from low to high were noted in 2 of the 25 patients (8%). The remaining 14 (56%) showed no evidence of changes in the DNA ploidy pattern. Reduction of DNA ploidy seen in HCC and its metastatic lesions in the lung may be one of the aspects of clonal evolution or selection mechanisms during the progression of tumor growth and metastasis.


The usefulness of the dynamic phase in pertechnetate thyroid imaging for solitary hypofunctioning nodules. One hundred patients, each with a solitary thyroid nodule detected by clinical palpation, underwent three-phase Tc-99m pertechnetate thyroid imaging. The degree of perfusion of the thyroid nodule was classified as hypo-perfused, euperfused, or hyperperfused compared to the remainder of the gland by a consensus of three nuclear medicine physicians. The nodules were subsequently biopsied, and the degree of perfusion of the nodules was correlated with their histologic diagnosis. Twenty-two nodules were classified as hyperperfused, 64 as euperfused, and 14 as hypoperfused. Malignancy rates of the hyperperfused, euperfused, and hypoperfused nodules were 36%, 31% and 0%, respectively. This seems to indicate that malignant thyroid nodules demonstrate a degree of perfusion at least equal to or greater than the rest of the thyroid gland. Conversely, none of the hypoperfused nodules was found to be malignant. The perfusion phase of thyroid imaging may provide useful clinical information regarding possible malignancy of a thyroid nodule.


Isotope angiography and blood pool imaging as a procedure for assessing radiation-induced injuries to the hands. The authors discuss radioisotope angiography and blood pool imaging for assessing radiation-induced injuries to the hands and the deterioration or improvement in status during follow-up. Two industrial radiographers in two separate accidents were exposed to 22 Ci to 25 Ci of a Co-60 for 2 to 3 minutes each in February 1985. Subsequently, besides routine clinical examinations and other tests, they underwent a series of amputations and grafts in the digits of the hands. A first-pass study with Tc-99m sodium pertechnetate followed by delayed RBC labeled blood pool imaging was performed in September 1988 in both patients. Both had a reduced flow of tracer to the more affected hand and the affected digits. A repeat study performed in March 1989 revealed improvement in perfusion to the more affected hand in the first patient and no change in the second. Delayed blood pool imaging did not reveal any change. The improvement in flow also coincided with the reduction of pain in the affected digits of the first patient. The authors conclude that isotope angiography followed by delayed blood pool imaging is a simple, noninvasive procedure to assess radiation-induced damage to extremities and to evaluate deterioration or improvement during follow-up.


Cytomegalovirus genomes demonstrated by polymerase chain reaction in synovial fluid from rheumatoid arthritis patients. Synovial fluid from 20 patients with rheumatoid arthritis was analyzed to detect human cytomegalovirus (CMV) genomic material using polymerase chain reaction. Of 20 samples tested, 9 were positive for CMV by either ethidium bromide staining or Southern blotting. In contrast, CMV was detected in only 2 of 18 control patients with osteoarthritis. These results suggest an etiologic relationship between CMV and rheumatoid arthritis.


Lipid peroxidation in experimental uveitis: sequential studies. Previously we have detected the occurrence of retinal lipid peroxidation initiated by phagocyte-derived oxygen radicals in experimental autoimmune uveitis (EAU). In the current studies, the confirmation of inflammation-mediated lipid peroxidation was proceeded further to include measurement of multiple parameters, including conjugated dienes, ketodienes, thiobarbituric acid reactive substances and fluorescent chromolipids. The assay for myeloperoxidase, a measure for the number of polymorphonuclear leukocytes in the inflammatory sites was also carried out. The levels of all these parameters were followed through the course of EAU development. The sequential evaluation of histologic changes using both light and electron microscopy was also carried out and the results were correlated with lipid peroxidation indices. These data suggest that the retinal lipid peroxidation plays a causative role in the subsequent retinal degeneration.


Modulation of immune responses by cyclo-oxygenase inhibitors during intraocular inflammation. The effects of cyclooxygenase inhibitors flurbiprofen and indomethacin on the inflammatory response and immune reactions induced by S-antigen in the rat eye were studied. The treatment with flurbiprofen and indomethacin administered subcutaneously every 12 hours, commenced on the day of S-antigen injection and was continued until the termination of the experiment (12 days). Flurbiprofen reduced vasodilation and the inflammatory exudate into the anterior chamber by 38% and 36% respectively while inhibiting polymorphonuclear leukocytes infiltration by 57% without affecting monocytes infiltration. Indomethacin had similar but greater effects than flurbiprofen on all these parameters. However, the differences between the mean values of the two compounds were not significant. Both compounds also attenuated spleen cell proliferation, serum IgG levels to S-antigen and interferon-gamma levels in the aqueous humor. The level of prostaglandin E2, but not of leukotriene B4, was increased in the untreated inflamed uveal tissues and this increase was significantly inhibited by flurbiprofen and indomethacin. The results of this study suggest that cyclooxygenase products are involved in the normal development of both humoral and cellular immune response in the experimental uveitis.


Assignment of the human urokinase receptor gene (PLAUR) to 19q13. Through in situ hybridization of a cDNA probe to metaphase chromosomes, we localized the gene for the human urokinase receptor (PLAUR) on chromosome 19. RBG-banding permitted subchromosomal localization of the PLAUR gene to 19q13.


Assignment of the human connexin 32 gene (GJB1) to band Xq13. The chromosomal localization of the human gene coding for connexin 32 (GJB1) was determined by in situ suppression hybridization (ISSH). The results allowed assignment of the gene to band Xq13, thus refining previous localization data obtained by means of somatic cell hybrid analysis.


The KDR gene maps to human chromosome 4q31.2----q32, a locus which is distinct from locations for other type III growth factor receptor tyrosine kinases. KDR (kinase insert domain receptor), a new type III receptor tyrosine kinase gene, maps to human chromosome 4q31.2----q32 by fluorescence in situ hybridization. This differs from the chromosomal locations of other members of this gene family.


A reciprocal whole-arm translocation, rcp(1;6)(1p6p;1q6q) in a boar, localization of the breakpoints, and reassignment of the genes for glucose phosphate isomerase (GPI) and calcium release channel (CRC). A reciprocal whole-arm translocation between chromosomes 1 and 6 in a Swiss Large White boar with reduced fertility was identified by the use of different staining techniques in mitotic metaphase cells, synaptonemal complex analyses, and meiotic chromosome preparations. The karyotype of this boar was demonstrated to be 38,XY,rcp(1;6)(1p6p;1q6q). To further localize the breakpoints more precisely and determine the precise gene locations, several in situ hybridization experiments were performed with a chromosome 1 centromere-specific probe and two other gene probes. The breakage and reunion points of both chromosomes were located in the centromeric regions. The genes for glucose phosphate isomerase and calcium release channel were mapped to 6cen----q12.


Protective effect of dimethylthiourea against mucosal injury in rat stomach. Implications for hydroxyl radical mechanism. The present study was undertaken to determine whether dimethylthiourea (DMTU), a hydroxyl radical scavenger, could prevent gastric injury in the rat stomach induced by various noxious agents. Fasted rats (N = 6-8/group) were given a 1-ml oral bolus of saline or DMTU over the dose range 10-500 mg/kg. After 30 min, animals received 1 ml of 100% ethanol orally and were sacrificed 5 min later. At sacrifice, stomachs were harvested and the degree of macroscopic damage was assessed by planimetry. In selected animals, specimens of gastric mucosa were also processed for histology. Saline pretreatment prior to ethanol exposure resulted in 22.5% injury to the glandular epithelium when assessed macroscopically. DMTU pretreatment prevented such injury in a dose-related fashion with only 2% of the mucosa showing injury with a 500 mg/kg dose (P less than 0.01 vs control). Although the superficial injury involving surface mucous cells induced by ethanol was not altered by DMTU, the deep damage to gastric glands was almost completely prevented. Other experiments in which DMTU was given intraperitoneally demonstrated similar protective effects against ethanol injury. Additional studies showed that indomethacin did not prevent the protective effects of oral or intraperitoneal DMTU, excluding a role for endogenous prostaglandins, and that DMTU was equally protective when administered within minutes or as long as 2 hr prior to ethanol exposure. Furthermore, DMTU was also shown to be protective against gastric injury induced by concentrated acid or base. In in vitro studies in which hydroxyl radicals were actually generated, DMTU was noted to scavenge the hydroxyl radical in a dose-related fashion. The ability of DMTU to prevent gastric injury by three different damaging agents suggests that the hydroxyl radical may play a major role in the pathogenesis of such injury and that DMTU mediated its protective action by scavenging this radical species.


Gastrointestinal transit times of radiolabeled meal in progressive systemic sclerosis. Gastrointestinal transit times were measured in 12 patients with progressive systemic sclerosis. The CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) was found in all patients. None of the patients reported complaints referable to specific gastric, small intestinal, or colonic involvement. The patient group had an increased mean gastric emptying time of 99mTc-labeled cellulose fiber when compared with 16 healthy controls [1.17 (0.89-1.38) hr [median (range)] vs 0.84 (0.56-1.88) hr; P less than 0.02], whereas mean gastric emptying time of 2- to 3-mm 111In-labeled plastic particles was unaffected [1.86 (0.99-2.74) hr vs 1.50 (0.92-2.51) hr; NS]. No difference was observed in mean small intestinal transit time of cellulose fiber [4.33 (0.50-7.04) hr vs 3.74 (2.09-7.59) hr; NS] or plastic particles [4.21 (2.00-6.25) hr vs 3.53 (1.50-6.70) hr; NS] between patients and controls. The patient group had an increased mean colonic transit time of plastic particles [47 (24-116) hr vs 29 (18-46) hr; P less than 0.01]. These findings suggest that asymptomatic delay in gastric emptying and colonic transit is frequent in patients with progressive systemic sclerosis.


MPO activity and generation of active O2 species in leukocytes from poorly controlled diabetic patients. This study was undertaken to determine which part of ROI generation is reduced in the neutrophils from patients with NIDDM. Superoxide anion (O2-) production, LDCL activity in response to opsonized zymosan, and MPO activity were measured in leukocytes of poorly controlled NIDDM patients (FBG greater than 8.89 mM, HbA1 greater than 10%). In diabetic subjects, both O2- production and LDCL activity assessed with initial slope gradient and peak value were significantly reduced. MPO activity was also decreased in diabetic subjects, and there was a significant correlation between HbA1 levels and MPO activity of diabetic subjects. This study demonstrated that every step in leukocyte ROI generation should be reduced in the leukocytes from poorly controlled diabetic patients.


Trends in diabetes and diabetic complications, 1980-1987. Although diabetes is a major source of morbidity and mortality in the United States, only recently has a unified national surveillance system begun to monitor trends in diabetes and diabetic complications. We established a diabetes surveillance system using data for 1980-1987 from vital records, the National Health Interview Survey, the National Hospital Discharge Survey, and the Health Care Financing Administration's records to examine trends in the prevalence and incidence of diabetes, diabetes mortality, hospitalizations, and diabetic complications. From 1980 through 1987, the number of individuals known to have diabetes increased by 1 million--to 6.82 million. Age-standardized prevalence for diabetes increased 9% during this period, from 25.4 to 27.6/1000 U.S. residents (P = 0.03). The incidence of diabetes increased among women (P = 0.003), particularly among those greater than 65 yr old (P = 0.02). Age-standardized mortality rates (for diabetes as either an underlying or contributing cause) per 100,000 individuals with diabetes declined 12%, from 2350 to 2066. Annual mortality rates from stroke (as an underlying cause and diabetes as a contributing cause) and diabetic ketoacidosis declined 29% (P = 0.003) and 22% (P less than 0.001), respectively. During these 8 yr, hospitalization rates for major CVD and stroke (as the primary diagnoses and diabetes as a secondary diagnosis) increased 34% (P = 0.006) and 38% (P = 0.01), respectively. Also during this period, hospitalization rates increased 21% for diabetic ketoacidosis (P = 0.01) and 29% for lower-extremity amputations (P = 0.06). From 1982 through 1986, treatment for end-stage renal disease related to diabetes increased greater than 10% each year (P less than 0.001). The prevalence of diagnosed diabetes was nearly twice as high in blacks as in whites (P = 0.04). Blacks also had increased rates of lower-extremity amputation (P = 0.02), diabetic ketoacidosis (P less than 0.001), and end-stage renal disease (P = 0.01). Diabetes surveillance data will be useful in planning, targeting, and evaluating public health efforts designed to prevent and control diabetes and its complications.


[Disulfiram neuropathy].

[Disulfiram neuropathy]. Two chronically alcohol-addicted patients, a 37-year old woman and a 22-year old man, developed a severe polyneuropathy after being treated with very high doses of disulfiram. In the first case there was a high-grade proximal tetraparesis, especially of the legs; in the other there was a similar largely motor, but predominantly distal, neuropathy. Electrophysiological tests established a pattern of axonal damage. Guillain-Barré syndrome was excluded by analysis of cerebrospinal fluid. Over a period of observation of 6 months and 2 1/2 years, respectively, the paresis continued even after disulfiram had been discontinued. 2 1/2 years later the electrophysiological changes were still present. As disulfiram treatment may cause severe side effects it is recommended that the lowest possible dosage by employed.


Posttranscriptional regulation of the human prolactin gene in IM-9-P3 cells by retinoic acid. The IM-9-P3 family of cell lines, which are derived from the B-lymphoblastoid IM-9 cell line, transcribe the human PRL (hPRL) gene by utilization of the decidual-type promoter and provide a model to study factors controlling extrapituitary expression of the hPRL gene. Here we describe regulation of hPRL gene expression in members of the IM-9-P3 family by retinoic acid (RA). When cells were incubated in medium supplemented with fetal calf serum that had been treated with dextran-coated charcoal, the addition of RA caused a 2-fold stimulation of hPRL secretion in the low hPRL-producing clone IM-9-P31 and the moderate producer IM-9-P32 (ED50, 0.53 and 0.13 nM, respectively), but not in the high hPRL-producing IM-9-P33 clone. Secretion from the RA-responsive cell lines increased steadily over the first 24 h of exposure and remained elevated for several days. The concomitant increase in hPRL mRNA steady state levels was not due to enhanced transcription of the hPRL gene, as assessed by nuclear run-on experiments, but, rather, to message stabilization. In RA-treated IM-9-P32 cells, the half-life of hPRL mRNA was significantly increased from 9 to 22 h. The transcripts were found to be preferentially associated with membrane-bound polysomes, thus being available for the secretory pathway. When we studied the expression of potential transducers of the RA signal, namely the RA receptor subtypes hRAR alpha, -beta, and -gamma and cellular RA-binding protein, we did not detect hRAR gamma or cellular RA-binding protein transcripts in the hPRL-negative clone IM-9-P6 or the hPRL-positive clones IM-9-P31, IM-9-P32, and IM-9-P33. hRAR alpha was equally expressed in all cell lines and not regulated by RA, whereas hRAR beta was differentially expressed and controlled by RA. This receptor subtype was absent from hPRL-negative members of the IM-9-P family, strongly induced by RA in the RA-responsive IM-9-P31 and IM-9-P32 cell lines via rapid transcriptional up-regulation, and only slightly induced in the RA-resistant IM-9-P33 cell line, suggesting a function in mediation of the effect of RA on hPRL gene expression.


Follicle-stimulating hormone induces functional receptors for basic fibroblast growth factor in rat granulosa cells. In the present study we examined the existence of receptors for basic fibroblast growth factor (bFGF) and its regulation in rat granulosa cells. The binding of labeled bFGF to rat granulosa cells was dose-dependently displaced by unlabeled bFGF, but not other growth factors. FSH induced a dose-dependent increase in specific binding for bFGF to cultured rat granulosa cells. FSH treatment did not change the binding affinity (Kd, 2.8-3.0 x 10(-10) M) of the bFGF receptor, but increased the total number of bFGF-binding sites, whereas treatment with several steroid hormones had no effect on the specific binding of bFGF. Since cycloheximide, a protein synthesis inhibitor, inhibited the increase in bFGF binding induced by FSH, it is suggested that protein synthesis might be involved in the FSH stimulation of bFGF receptor induction. Furthermore, bFGF stimulated tissue plasminogen activator activity in a dose-dependent manner, and FSH-primed granulosa cells were more responsive to bFGF action, with a decrease in the ED50 from 5.0 to 1.5 ng/ml. The antibody against human bFGF neutralized the stimulatory effect of bFGF on tissue plasminogen activator secretion. The present study suggests that FSH induces functional receptors for bFGF in granulosa cells and that bFGF may play a role in the process of differentiation under the influence of FSH.


Pituitary adenylate cyclase-activating polypeptide stimulates calcium mobilization in amphibian pituitary cells. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily. Although PACAP is a potent stimulator of adenylate cyclase activity in the adenohypophysis, the precise target cells for PACAP in the anterior pituitary remain unknown. The aim of the present study was to investigate whether PACAP could stimulate calcium mobilization in individual cells of the pituitary and to determine the type of cells that responded to PACAP. Enzymatically dispersed frog distal pituitary cells were plated on photoetched coverslips and cultured for 3-7 days. The cells were loaded with the fluorescent calcium indicator indo-1, and changes in intracellular calcium concentrations ([Ca2+]i) were monitored using dual wavelength microfluorimetry. The individual cells were localized with the aid of the alpha/numeric grid of the coverslips and identified retrospectively by immunofluorescence. Approximately 45% of GH and PRL cells and 25% of ACTH and TSH cells responded to PACAP (10(-5) M) ejection by an elevation of [Ca2+]i. Only 16% of gonadotropes were stimulated by PACAP. The time course of [Ca2+]i variations showed three different patterns: transient spikes, sustained stimulations, and oscillatory responses. In addition, heterogenous responses were observed within each cell type. These data provide evidence for the involvement of calcium mobilization in the mechanism of action of PACAP on pituitary cells. The results also indicate that in frogs, PACAP may stimulate the secretory activity of GH and PRL cells and, to a lesser extent, ACTH, TSH, and gonadotrope cells.


Identification of endothelin receptor subtypes in human renal cortex and medulla using subtype-selective ligands. High affinity and high density endothelin (ET)-binding sites were identified in membranes prepared from human kidney cortex and medulla. Saturation binding experiments performed in membranes prepared from cortex and medulla using [125I]ET-1 and [125I]ET-3 revealed that the proportion of [125I]ET-3-binding sites was 30-35% less than that of [125I]ET-1-binding sites. The apparent dissociation constants and maximum binding for [125I]ET-1 and [125I]ET-3 to membranes from cortex were 91 +/- 5 pM and 165 +/- 10 fmol/mg protein, and 117 +/- 9 pM and 110 +/- 7 fmol/mg protein, respectively, whereas in medulla they were 139 +/- 10 pM and 360 +/- 11 fmol/mg protein, and 142 +/- 11 pM and 245 +/- 15 fmol/mg protein, respectively. In the presence of 10 nM sarafotoxin-6c, which is selective for ETB receptors, [125I]ET-1 binding was decreased by 65-70%, whereas [125I]ET-3 binding was totally abolished, suggesting that 65-70% of [125I]ET-1 binding and 100% of [125I]ET-3 binding was to ETB receptors. This was further confirmed by the use of a cyclic pentapeptide [cyclo(D-Trp,D-Asp,L-Pro, D-Val,L-Leu)] (BQ123), which is selective for ETA receptors. In the presence of 1 microM BQ123, [125I]ET-1 binding was decreased by 25-30%, whereas [125I]ET-3 binding was unaffected, confirming that 30-35% of ET receptors belong to the ETA subtypes, and that [125I]ET-1 bound to both ETA and ETB receptors with the same high affinity, but [125I]ET-3 bound only to ETB receptors with high affinity. These results suggest that human kidney cortex and medulla contain ETA and ETB receptors in a ratio of 30:70, and that sarafotoxin-6c and BQ123 are valuable tools in identifying the subtype of ET receptors in various tissues.


Hypothalamic paraventricular nuclear lesions delay corticotroph maturation in the fetal sheep anterior pituitary. The purpose of this study was to determine whether normal morphological development occurs in pituitary corticotrophs deprived of products of the hypothalamic paraventricular nucleus (PVN), e.g. corticotropin releasing hormone and arginine vasopressin (AVP), after PVN lesions. In addition, we have attempted to ascertain if the neurophysin/AVP-positive fibers innervating the fetal sheep anterior pituitary are affected by PVN lesions. The experimental groups consisted of fetal sheep in which 1) hypothalamic PVN lesions were placed at 118-122 days gestation (dGA) and the fetuses subsequently harvested while still in utero at 157 dGA or more (PVNX; n = 5); 2) sham PVN lesions were placed at 118-122 dGA and subsequently harvested as newborn lambs immediately after birth at 146.5 +/- 0.9 (mean +/- SEM) dGA combined with two uninstrumented fetuses harvested at 144 dGA or more but not in labor (perinatal; n = 6); and 3) no instrumentation was placed, and the fetuses were harvested at 120 dGA (control; n = 4). Two ACTH-immunoreactive cell types were seen in the anterior pituitary: 1) fetal cells: large and variably stained, often columnar, occurring in clusters and arranged in palisades; and 2) adult cells: smaller, darkly staining, and angular, occurring singly or in small groups. Quantification of the distribution of the two ACTH cell types was performed by scanning sections from a one in six series from each pituitary and estimating the percent area of each section in the well that showed adult type staining only. The observer was blind to the treatment group assignment of the sections. The estimated percentages of the portion of the pituitaries of each group that contained adult-type cells only were as follows: PVNX, 42.8 +/- 10.0%; perinatal, 90.9 +/- 2.1%; and control, 3.7 +/- 1.1% (mean +/- SEM; P less than 0.05 for all comparisons). There were no qualitative differences between all groups in the appearance of neurophysin-positive fibers innervating the anterior pituitary. AVP staining was strong in the internal zone of the median eminence in all groups, but was absent in the external zone of PVNX fetuses only. The intermediate pituitary lobes stained darkly in all groups. We conclude that lesions of the PVN at 120 dGA delay development of fetal pituitary corticotrophs, but have no effect on the presence of neurophysin-positive nerve fibers in the anterior pituitary.


1,25-dihydroxyvitamin D3 inhibits Na(+)-H+ exchange by stimulating membrane phosphoinositide turnover and increasing cytosolic calcium in CaCo-2 cells. We have examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the phosphoinositol signal transduction pathway in the human colon cancer-derived cell line CaCo-2 and have studied the regulation of intracellular calcium ([Ca2+]i) and pH (pHi) by this secosteroid. CaCo-2 cells were prelabeled with [3H]myoinositol and treated with 10(-8) M 1,25-(OH)2D3 or vehicle for 90 sec. 1,25-(OH)2D3 caused a decrease in labeled phosphatidylinositol-4-5-bis-phosphate and an increase in labeled inositol 1,4,5-trisphosphate. Treatment with 10(-8) M 1,25-(OH)2D3 for 90 sec also raised the cellular content of diacylglycerol. In a dose-dependent manner, 1,25-(OH)2D3 caused the translocation of protein kinase-C activity from the cytosolic to the membrane fraction, which occurred after as little as 15 sec of exposure to the secosteroid, peaked at about 1-5 min, and then returned toward baseline values. In these CaCo-2 cells, baseline [Ca2+]i was 258 +/- 2 nM (mean +/- SE), as assessed using the fluorescent dye fura-2. After exposure to 10(-8) M 1,25-(OH)2D3, [Ca2+]i rapidly increased to 392 +/- 14 nM after 100 sec, fell, and then subsequently rose to a plateau of 350 +/- 3 nM after 400 sec. In Ca(2+)-free buffer, 1,25-(OH)2D3 caused only a transient rise in [Ca2+]i, indicating that 1,25-(OH)2D3 stimulated both the release of intracellular calcium stores and calcium influx. 1,25-(OH)2D3 caused a dose-dependent decrease in pHi in CaCo-2 cells, as assessed by the fluorescent dye BCECF, which was not observed in cells suspended in Na(+)-free buffer or pretreated with amiloride, indicating that the secosteroid inhibited Na(+)-H+ exchange. No effect of 1,25-(OH)2D3 on pHi was observed in cells in a Ca(2+)-free buffer or pretreated with the phospholipase-C inhibitor U-73,122, which also blocked the rise in [Ca2+]i, or in cells pretreated with the Ca2+/calmodulin inhibitor calmidazolium. Taken together, these studies indicate that 1,25-(OH)2D3 rapidly stimulates membrane phosphoinositide breakdown in CaCo-2 cells, generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, causing translocation of protein kinase-C to the membrane, and increasing [Ca2+]i by both releasing calcium stores and promoting calcium influx. Secondary to the rise in [Ca2+]i, Na(+)-H+ exchange is inhibited by a calcium/calmodulin-dependent pathway.


Comparison of the effects of the 5 alpha-reductase inhibitor finasteride and the antiandrogen flutamide on prostate and genital differentiation: dose-response studies. Studies were performed to compare the effects of 5 alpha-reductase inhibition and antiandrogen receptor blockade on differentiation of male internal and external genital structures and prostate in the rat. Dose-response studies were performed on male rats treated in utero during the period of sexual differentiation with either the potent 5 alpha-reductase inhibitor finasteride or the antiandrogen flutamide. The treated animals were raised to adulthood and killed, and genital structures were evaluated. Treatment with the 5 alpha-reductase inhibitor finasteride at a dose of 25 mg/kg.day resulted in significant feminization of the external genitalia. There was no further feminization of the genitalia at doses up to 300 mg/kg.day. Wolffian ductal differentiation occurred at all doses evaluated. Seminal vesicle weight, however, significantly decreased at 25 mg/kg.day, but without a further decrease at higher doses of the 5 alpha-reductase inhibitor. Vas deferens and epididymal weights were unchanged at all doses evaluated. There was a significant decrease in prostate size at 25 and 50 mg/kg.day, with no further decrease at higher doses. In flutamide-treated animals, complete feminization of the genitalia occurred at 24 mg/kg.day in all animals. At 18 mg/kg.day, Wolffian ductal differentiation occurred, but seminal vesicle weight was decreased. At dosages of 100, 200, and 300 mg/kg.day flutamide, the vas deferens was absent unilaterally or bilaterally, with small remnants of epididymal head and tail present. At dosages of 24 mg/kg.day and above, the prostate was absent. Studies with the 5 alpha-reductase inhibitor finasteride demonstrate the dependency of prostate and male external genital differentiation on dihydrotestosterone (DHT). However, unlike androgen receptor blockade with flutamide, finasteride did not totally abolish prostate differentiation or completely feminize the external genitalia, despite increasingly higher doses. Since there is no evidence of multiple 5 alpha-reductase isoenzymes to date in the rat, these results suggest that testosterone (T) can compensate for DHT to some degree at the level of the androgen receptor. Wolffian differentiation, however, was not affected by inhibition of DHT, demonstrating its T dependency, but seminal vesicle growth was impaired. Thus, inhibition of 5 alpha-reductase activity limits seminal growth potential in adulthood. Studies with the antiandrogen flutamide show that at doses significantly above that required to completely block prostate differentiation and cause genital feminization, Wolffian ductal differentiation is significantly impaired. Thus, higher doses of flutamide are needed to block the paracrine effect of T on the Wolffian ducts.


Effects of vasoactive intestinal peptide on adenosine 3',5'-monophosphate, ornithine decarboxylase, and cell growth in a human colon cell line. Vasoactive intestinal peptide (VIP) is a widely distributed neuropeptide that has been considered a potential regulator of cell growth and differentiation in various tissues, including the gut. To examine this idea, we used a human colon carcinoma cell line (LoVo) as a model system and measured ornithine decarboxylase (ODC), because this is the rate-limiting enzyme for the formation of polyamines, which are thought to be key factors in regulating cell growth. LoVo cells, grown to about 80% confluence in F-12 medium containing 10% fetal bovine serum, were preincubated for 5 h in low serum medium (1% fetal bovine serum in F-12), and ODC activity was determined by measuring 14CO2 liberated from 14C-labeled ornithine. VIP caused a dose-related biphasic change in ODC, with activity increased at 10 pM, maximal (5-fold increase) at 10 nM, and decreased toward basal at 100 nM to 1 microM. Incubation of cells for 6 days with VIP in low serum medium showed similar changes in cell numbers, with growth being increased by doses in the 1 pM to 100 nM range and decreased at higher doses (greater than or equal to 100 nM). Exposure of cells to 5 mM alpha-difluoromethylornithine blocked both the VIP-induced increase in cell number and the VIP-induced increase in ODC activity. Increased ODC mRNA was detected after 2 h of exposure to VIP, a time at which ODC activity peaked after treatment, and the increase in ODC mRNA caused by VIP was dose-dependent. In related experiments LoVo cells were found to have high affinity VIP receptors (Kd = 0.4 nM), as assessed by examination of [125I]VIP binding in the presence of varying concentrations of unlabeled VIP. Studies of intracellular cAMP revealed a dose-related increase in cAMP in response to VIP (ED50 = 11 pM), and the adenylate cyclase activator forskolin increased both ODC activity and ODC mRNA. The findings support the idea that LoVo cells have VIP receptors linked to cAMP which can stimulate cell growth at least in part by increasing ODC synthesis and activity, thereby altering the production of polyamines. The decreased growth and ODC activity observed with high doses of VIP may involve a second messenger other than cAMP.


Expression of the insulin-like growth factor (IGF)-I and -II and the IGF-I and -II receptor genes during postnatal development of the rat ovary. Solution hybridization/RNase protection assays were used to study the developmental expression of the insulin-like growth factor-I (IGF-I), IGF-II, IGF-I receptor, and IGF-II/mannose-6-phosphate receptor genes in the rat ovary between postnatal days 1-80. Maximal IGF-I mRNA levels occurred during the 15- to 25-day postnatal period, and the level on day 20 represented a 9-fold increase over the baseline at earlier and later stages. IGF-II mRNA levels were maximal during the 1- to 5-day postnatal period and subsequently declined to undetectable levels after day 10. IGF-I receptor mRNA levels increased 10-fold to a maximum in the 20- to 25-day postnatal period. This pattern was similar to the developmental pattern of [125I]IGF-I binding in the ovary. Two apparent peaks of IGF-II/mannose-6-phosphate receptor mRNA levels were seen, on day 20 and between days 50-80. These specific and significant changes in the expression of the genes encoding the IGFs and their receptors suggest a role for the IGF system in postnatal ovarian development.


Chronic estrogen-induced alterations in adrenocorticotropin and corticosterone secretion, and glucocorticoid receptor-mediated functions in female rats. The effect of estrogen (E) on the hypothalamic-pituitary-adrenal axis was investigated in female Sprague-Dawley rats. Animals were bilaterally ovariectomized (OVX), and a Silastic capsule (0.5 cm) containing 17 beta-estradiol was sc implanted. Control animals received a blank capsule. Animals were killed 21 days later. In E-treated rats, we found significantly higher corticosterone (CORT) peak levels 20 min after a 5-sec footshock (1.0 mamp) or exposure to ether vapors (P less than 0.05) compared to those in OVX controls. In addition, the recovery of the ACTH and CORT responses to footshock stress was significantly prolonged (P less than 0.05) in the presence of E. Furthermore, the ACTH and CORT secretory responses to ether stress could be suppressed by exogenous RU 28362 (a specific glucocorticoid receptor agonist; 40 micrograms/100 g BW for 4 days) in OVX controls (P less than 0.05), but not in E-treated animals. These data suggest that E can impair glucocorticoid receptor-mediated delayed or slow negative feedback. Consequently, we examined the influence of E on mineralocorticoid and glucocorticoid receptor concentrations using in vitro binding assays. E did not alter mineralocorticoid or glucocorticoid receptor concentrations in any of the brain regions examined. The administration of RU 28362 (40 micrograms/100 g BW for 4 days) to OVX control or E-treated rats significantly down-regulated hippocampal glucocorticoid receptor (P less than 0.02) in control rats only. In contrast, aldosterone administration (40 micrograms/100 g BW for 4 days) significantly down-regulated hippocampal glucocorticoid receptor (P less than 0.0008) in both control and E-treated animals. Thus, E treatment results in a loss of the glucocorticoid receptor's ability to autoregulate; this suggests that E may cause a functional impairment of the glucocorticoid receptor even though receptor binding appears normal. These findings suggest that hyperactivation of the hypothalamic-pituitary-adrenal axis after stress in E-treated rats is due in part to impaired glucocorticoid receptor-mediated slow negative feedback.


Thyroid hormone receptors and 3,5,3'-triiodothyronine biological effects in FRTL5 thyroid follicular cells. Specific thyroid hormone (T3) receptors are present in thyroid follicular cells, including the rat FRTL5 clonal line, but little is known about the effects of T3 on the growth and differentiated function of the thyroid. Unlike primary cultures of animal or human thyroid cells, FRTL5 do not secrete appreciable amounts of thyroid hormones. We now have studied the effects of T3 by itself and in combination with TSH and insulin-like growth factor-I (IGF-I) on [3H]thymidine incorporation into DNA, iodide uptake, and cAMP production in FRTL5. We also have investigated the expression of different c-erbA mRNAs in these cells. Specific binding of T3 to FRTL5 cell nuclei in intact cells occurred with a binding capacity of 0.1-0.15 ng T3/mg DNA and an apparent Kd of 0.4 nM. Using an RNase protection assay on total cellular FRTL5 RNA and specific cRNA probes, we demonstrated the presence of c-erbA alpha and -beta mRNAs, both encoding T3 receptors. Biological effects were assessed in serum-free medium or buffer containing 0.1% BSA after maintaining quiescent culture of cells for at least 5 days in hormone-free medium containing 5% calf serum. T3 alone stimulated a dose-dependent increase in [3H]thymidine incorporation that reached a plateau at 188% of the control value at 10 nM T3. At 10(-11) M TSH, T3 potentiated TSH-stimulated [3H]thymidine incorporation (2.2-fold), but at TSH concentrations greater than 5 x 10(-11) M, T3 had no effect or reduced the response to TSH. T3 potentiated the [3H]thymidine response to 2 and 10 ng/ml IGF-I by 1.5- to 1.7-fold. T3 alone had no effect on iodide uptake, but attenuated iodide uptake stimulated by TSH. T3 was more potent in inhibiting TSH-stimulated iodide uptake than in enhancing TSH-stimulated DNA synthesis. T3 did not affect either basal or TSH-stimulated cAMP accumulation. Thus, in FRTL5 thyroid follicular cells 1) T3 receptors are expressed, as measured by direct binding assays and by the expression of c-erbA mRNAs; and 2) T3 acts as a growth factor and weak antidifferentiation factor. We suggest that T3 may modulate the actions of TSH and growth factors in thyroid epithelium.