Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN).

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.

Role of tyrosine phosphorylation in thrombin-induced endothelial cell contraction and barrier function.

Thrombin-induced endothelial cell (EC) barrier dysfunction is highly dependent upon phosphorylation of serine and threonine residues present on myosin light chains (MLC) catalyzed by a novel EC myosin light chain kinase (MLCK) isoform. In this study, we examined the participation of tyrosine protein phosphorylation in EC contraction, gap formation and barrier dysfunction. We first determined that thrombin significantly increases protein tyrosine kinase activity and protein tyrosine phosphorylation in bovine pulmonary artery EC. Tyrosine kinase inhibitors, genistein and 2,5 DHC, reduced EC tyrosine kinase activities, however, only genistein significantly attenuated thrombin-mediated increases in albumin clearance and reductions in transendothelial electrical resistance. Similarly, genistein but not 2,5 DHC, decreased basal and thrombin-induced Ca2+ increases and MLC phosphorylation in the absence of alterations in Type 1 or 2A serine/threonine phosphatase activities. Immunoprecipitation of the EC MLCK isoform revealed a 214 kD immunoreactive phosphotyrosine protein and genistein pretreatment significantly reduced MLCK activity in MLCK immunoprecipitates. Although thrombin induced the translocation of p60src from the cytosol to the EC cytoskeleton, a detectable increase in the level of MLCK tyrosine phosphorylation was not noted after thrombin challenge. Taken together, our data suggest that genistein-sensitive tyrosine kinase activities are involved in thrombin-mediated EC MLCK activation, MLC phosphorylation, and barrier dysfunction.

Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores.

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of Gs. Measurements of intracellular free calcium concentration ([Ca2+]i) reveal that mastoparan induces a dramatic elevation of [Ca2+]i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca2+]i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca2+]i is maintained in the absence of extracellular Ca2+, suggesting that the rise of [Ca2+]i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca2+]i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca2+]i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca2+]i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca2+]i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca2+ release from intracellular stores, probably via activation of PLC and IP3. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.

Pregnenolone sulfate augments NMDA receptor mediated increases in intracellular Ca2+ in cultured rat hippocampal neurons.

The ability of the neuroactive steroid pregnenolone sulfate to alter N-methyl-D-aspartate (NMDA) receptor-mediated elevations in intracellular Ca2+ ([Ca2+]i) was studied in cultured fetal rat hippocampal neurons using microspectrofluorimetry and the Ca2+ sensitive indicator fura-2. Pregnenolone sulfate (5-250 microM) caused a concentration-dependent and reversible potentiation of the rise (up to approximately 800%) in [Ca2+]i induced by NMDA. In contrast, the steroid failed to alter basal (unstimulated) [Ca2+]i or to modify the rise in [Ca2+]i that occurs when hippocampal neurons are depolarized by high K+ in the presence of the NMDA receptor antagonist CPP. These data suggest that the previously reported excitatory properties of pregnenolone sulfate may be due, in part, to an augmentation of the action of glutamic acid at the NMDA receptor.

Pharmacodynamics of the hypotensive effect of levodopa in parkinsonian patients.

Blood pressure effects of i.v. levodopa were examined in parkinsonian patients with stable and fluctuating responses to levodopa. The magnitude of the hypotensive effect of levodopa was concentration dependent and was fit to an Emax model in fluctuating responders. Stable responders demonstrated a small hypotensive response. Baseline blood pressures were higher in fluctuating patients; a higher baseline blood pressure correlated with greater hypotensive effects. Antiparkinsonian effects of levodopa temporally correlated with blood pressure changes. Phenylalanine, a large neutral amino acid (LNAA) competing with levodopa for transport across the blood-brain barrier, reduced the hypotensive and antiparkinsonian effects of levodopa. We conclude that levodopa has a central hypotensive action that parallels the motor effects in fluctuating patients. The hypotensive effect appears to be related to the higher baseline blood pressure we observed in fluctuating patients relative to stable patients.

Minor analgesic use among high-school students: indications for renal morbidity and mortality.

Reported patterns of minor analgesic use by adolescents were taken from a baseline 1973 survey, and a follow-up 1974 survey. The differences between the sexes within each year were shown to be highly significant. Trend analysis in minor analgesic use included the year-to-year comparisons, the degree of minor analgesic use, and the rate of change in this use. The trends show the long-term process of minor analgesic use, and support the cause for concern about analgesic nephropathy.

From Ottawa, Uppsala, and Alma-Ata to Canberra, Australia: a rationale for a degree in health.

The paper describes the principles of curriculum design applied in establishing the Degree of Bachelor of Applied Science in Health Education in Canberra, Australia in 1979. The design was based explicitly on three major initiatives in health planning and policy in the last decade: (i) the recommendations to the Canadian Government proposed by Lalonde in 1974, commonly called the Health Field Concept; (ii) the World Health Organization definition of Health, first stated in 1948, and reissued at Uppsala in 1977; and (iii) the World Health Organization policy statement from the Alma-Ata Seminar in 1978, which included social planning and legislative action among the legitimate concerns of health services. The authors, who are at present teaching the integrated units of the degree, describe the principal components, namely content, theoretical principles, professional skills and methods of knowledge integration which they are using to develop graduates who may be considered wither health educated or health educators. In either case, the program is intended to produce people who can provide a health development arm for, on the one hand, social planning and social change, and on the other, health-care services.

N-methyl-D-aspartate induces a rapid, reversible, and calcium-dependent intracellular acidosis in cultured fetal rat hippocampal neurons.

The ability of NMDA to alter intracellular pH (pHi) was studied in fetal rat hippocampal neurons and glia using the pH-sensitive fluorescent indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Brief exposure (60 sec) of hippocampal neurons to NMDA (2.5-250 microM) results in a rapid, and in most cells reversible, reduction in pHi, with full recovery to baseline pHi values taking several minutes following removal of NMDA. In contrast, little or no change in pHi was observed in glial cells exposed to these same concentrations of NMDA. The NMDA-induced acidification of neurons was concentration and time dependent, with an EC50 of 39 microM and Emax (delta pH) of -0.53. More prolonged exposure to NMDA (> or = 10 min) resulted in a more prolonged reduction in pHi values over the ensuing 20 min observation period. The intracellular acidification resulting from NMDA exposure of hippocampal neurons was blocked by the NMDA receptor antagonist 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). Moreover, removal of extracellular Ca2+ eliminated both the selective NMDA-induced elevation in [Ca2+]i and the reduction in pHi, indicating that Ca2+ influx may be required for the decrease in pHi induced by NMDA receptor activation. Finally, the NMDA-induced reduction in pHi was not significantly attenuated when extracellular [H+] was decreased by increasing extracellular pH to 8.0. The latter suggests that an intracellular source of H+ is responsible for the NMDA-induced reduction in neuronal pHi. The reduction in neuronal pHi induced by NMDA receptor activation may mediate some of the physiological and (or) pathophysiological actions of glutamate.

Characterization of the excitoprotective actions of N-methyl-D-aspartate in cultured cerebellar granule neurons.

Exposure of cultured cerebellar granule neurons to subtoxic concentrations of N-methyl-D-aspartate (NMDA) has been shown previously to result in a neuroprotective state, as measured by subsequent exposure to toxic concentrations of glutamate. In the present study, we have further characterized the excitoprotective actions of NMDA in these neurons. NMDA-induced excitoprotection was concentration dependent (EC50 approximately 30 microM) and time dependent, with maximal protection observed following 16 h of preexposure to NMDA. NMDA-induced excitoprotection did not require continuous exposure to NMDA, as a 4-h preincubation was sufficient to induce full excitoprotection when measured 8 h later. Maximal protection was manifest as a "right shift" in the concentration-response relationship for glutamate toxicity of approximately three orders of magnitude (EC50 approximately 30 microM in untreated neurons compared with > or = 50 mM in NMDA-treated neurons). After removal of NMDA, complete reversal of the excitoprotective state was observed by 48 h (t1/2 approximately 24 h). The ability of NMDA to induce excitoprotection was observed in neurons maintained for up to 14 days in vitro (DIV) [postnatal day (PND) 22], but was absent at 21 and 32 DIV (PND 29-40), despite little to no difference in the toxicity of glutamate at any DIV examined. Preexposure of cerebellar granule neurons to a maximally excitoprotective concentration of NMDA (50 microM) failed to alter the density of NMDA receptors measured by the specific binding of [3H]MK-801.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of muscarinic cholinergic receptors blocks apoptosis of cultured cerebellar granule neurons.

We have recently reported that the majority of cultured rat cerebellar granule neurons undergo apoptosis when maintained in the presence of physiological concentrations of K+ (nondepolarizing conditions). We now report that exposure of cultured cerebellar granule neurons, maintained under nondepolarizing conditions, to the muscarinic cholinergic receptor (mAchR) agonists carbachol and muscarine results in a concentration- and time-dependent inhibition of apoptosis. The nicotinic cholinergic receptor agonist (-)-nicotine fails to mimic, and the nicotinic cholinergic receptor antagonist dihydro-beta-erythroidine fails to antagonize, the survival-promoting effects of carbachol. In contrast, relatively low concentrations of atropine completely prevent the effects of carbachol in blocking apoptotic death of cultured granule neurons. Although the m1- and m2-preferring mAchR antagonists pirenzepine and gallamine, respectively, fail to reverse the effects of carbachol, the m3-preferring antagonist 4-diphenylacetoxyl-N- methylpiperidine methiodide completely blocks the survival-promoting effects of carbachol. These data demonstrate that activation of the mAchR (possibly of the m3 subtype) blocks apoptosis of cultured cerebellar granule neurons. The antiapoptotic effects of mAchR agonists are not indirectly mediated via glutamate release from granule neurons, because antagonists of either N-methyl-D-aspartate or non-N-methyl-D-aspartate glutamate receptors fail to affect the antiapoptotic effects of carbachol or muscarine. Moreover, exposure of cultured cerebellar granule neurons to antiapoptotic concentrations of carbachol, in contrast to high concentrations of K+ or glutamate receptor agonists, results in only a small and transient elevation of the intracellular Ca2+ concentration, as measured by fura-2 microfluorimetry. Slow neurotransmitters such as acetylcholine, acting via their cognate G protein-coupled receptors, may prevent neuronal apoptosis in the developing (and perhaps adult) central nervous system.

Activation of G proteins bidirectionally affects apoptosis of cultured cerebellar granule neurons.

Cultured cerebellar granule neurons maintained in depolarizing concentrations of K+ (25 mM) and then switched to physiological concentrations of K+ (5 mM) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apotosis of cultured rat cerebellar granule neurons. Stimulation of Gs with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K+, whereas stimulation of Go/Gi with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K+. Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell-permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS.

Steroid potentiation and inhibition of N-methyl-D-aspartate receptor-mediated intracellular Ca++ responses: structure-activity studies.

Pregnenolone sulfate and 15 related steroids were investigated for their effects on N-methyl-D-aspartate (NMDA)-induced elevations in intracellular Ca++ ([Ca++]i) in cultured rat hippocampal neurons by microspectrofluorimetry with the Ca(++)-sensitive indicator fura-2. Several pregn-5-ene steroids markedly potentiated NMDA-mediated [Ca++]i responses. Pregnenolone sulfate and its 21-acetoxy derivative and pregnenolone hemisuccinate were the most active. At a concentration of 50 microM, each produced approximately 300% potentiation of 5 microM NMDA responses. In addition, several steroids were identified that inhibited NMDA-induced elevations in [Ca++]i, the most potent of which was 3 alpha-hydroxy-5 beta-pregnan-20-one sulfate (IC50, 37 microM). Concentration-response curves for NMDA in the presence of active steroids revealed noncompetitive interaction(s) of these steroids with the NMDA receptor. Although the mechanism(s) responsible for either steroid-induced augmentation or inhibition of NMDA-receptor responses is unknown, these data suggest the presence of one or more steroid recognition sites with a high degree of structural specificity associated with NMDA receptors. These results further raise the possibility that pregn-5-ene 3-sulfates and pregnane 3-sulfates could be endogenous modulators of NMDA receptor-mediated synaptic events.

Regulation of endothelial cell myosin light chain phosphorylation and permeability by vanadate.

The involvement of tyrosine protein phosphorylation in the regulation of endothelial cell (EC) contraction and barrier function is poorly understood. We have previously shown that myosin light chain (MLC) phosphorylation catalyzed by a novel 214 kDa EC myosin light chain kinase (MLCK) isoform is a key event in EC contraction and barrier dysfunction [Garcia et al. (1995): J Cell Physiol 163:510-522; Garcia et al. (1997): Am J Respir Cell Mol Biol 16:487-491]. In this study, we tested the hypothesis that tyrosine phosphatases participate in the regulation of EC contraction and barrier function via modulation of MLCK activity. The tyrosine phosphatase inhibitor, sodium orthovanadate (vanadate), significantly decreased electrical resistance across bovine EC monolayers and increased albumin permeability consistent with EC barrier impairment. Vanadate significantly increased EC MLC phosphorylation in a time-dependent manner (maximal increase observed at 10 min) and augmented both the MLC phosphorylation and permeability responses produced by thrombin, an agonist which rapidly increases tyrosine kinase activities. The vanadate-mediated increase in MLC phosphorylation was not associated with alterations in either phosphorylase A Ser/Thr phosphatase activities or in cytosolic [Ca2+] but was strongly associated with significant increases in EC MLCK phosphotyrosine content. These data suggest that tyrosine phosphatase activities may participate in EC contractile and barrier responses via the regulation of the tyrosine phosphorylation status of EC MLCK.

Carbamazepine inhibition of N-methyl-D-aspartate-evoked calcium influx in rat cerebellar granule cells.

The effect of carbamazepine (CBZ) on N-methyl-D-aspartate (NMDA)-stimulated CA++ influx in rat cerebellar granule cells was studied by use of fura-2 microfluorometry. CBZ inhibited the rise in intracellular free Ca++ concentration ([Ca++]i) induced by NMDA and glycine in a rapid reversible and concentration-dependent manner. CBZ's inhibition of the [Ca++]i increase was noncompetitive with respect to NMDA, glycine and the facilitatory neurosteroid pregnenolone sulfate. The degree of inhibition of the NMDA response produced by CBZ increased with increasing concentrations of extracellular KCl. Excluding non-NMDA receptor-mediated contributions to Ca++ influx, depolarization by 50 mM KCl resulted in a 20-fold decrease (from 723 to 33 microM) in the IC50 for CBZ blockade of the NMDA response. Thus, significant blockade of NMDA receptor responses in cerebellar granule cells can occur at concentrations of CBZ within the therapeutic range under conditions believed to accompany seizures. Moreover, the common toxic side effects of CBZ, which include signs of cerebellar dysfunction, may occur as a result of CBZ blockade of the NMDA receptors of cerebellar granule cells.

Diphenylhydantoin induces apoptotic cell death of cultured rat cerebellar granule neurons.

Apoptosis is one form of physiological or programmed cell death responsible for the selective elimination of various cell types during development. We have observed and characterized a delayed-type of neurotoxicity induced in cultured cerebellar granule neurons by diphenylhydantoin. Diphenylhydantoin toxicity of cerebellar granule neurons is time and concentration dependent. Morphological studies using Nomarski optics and staining with the fluorescent dye Hoechst 33258 demonstrate that diphenylhydantoin-induced neurotoxicity of cerebellar granule neurons is associated with cytoplasmic blebbing, heterochromatic clumping and condensation of chromatin that precede cell death. Unlike glutamate toxicity (excitotoxicity) diphenylhydantoin-induced neurotoxicity of cerebellar granule neurons is attenuated by actinomycin D and cycloheximide, and is associated with nucleosomal size DNA fragmentation. Since we have previously reported that depolarization of cultured cerebellar granule neurons with high concentrations of K+ promotes the survival of these neurons by blocking apoptosis, we examined the effects of diphenylhydantoin on the K(+)-evoked increase in intracellular calcium. Using microfluorimetry and fura-2 to measure intracellular calcium we found that neurotoxic concentrations of diphenylhydantoin markedly reduce the increase in intracellular calcium associated with elevated extracellular potassium. Taken together, these data demonstrate that exposure of cultured cerebellar granule neurons to pharmacologically relevant concentrations of diphenylhydantoin results in a delayed type of neurotoxicity characterized by the biochemical and morphological features of apoptosis.