Robert P. Irwin

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.

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.

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.

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

Abstract: 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 apoptosis 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.

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. J. Cell. Biochem. 70:141–155, 1998.

Characterization of the Excitoprotective Actions of N‐Methyl‐d‐Aspartate in Cultured Cerebellar Granule Neurons

Abstract: 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∼30 µM) 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∼30 µM in untreated neurons compared with ≥50 mM in NMDA‐treated neurons). After removal of NMDA, complete reversal of the excitoprotective state was observed by 48 h (t1/2≈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 µM) failed to alter the density of NMDA receptors measured by the specific binding of [3H]MK‐801. Moreover, the immediate elevation in intracellular free calcium concentration ([Ca2+]i) induced by glutamate exposure and measured by microfluorimetry and the Ca2+‐sensitive indicator fura‐2 was similar in NMDA‐pretreated and untreated neurons. As reported previously, NMDA‐induced excitoprotection in cerebellar granule neurons was, however, reversed by coincubation with the protein synthesis inhibitor cycloheximide. Taken together, these data suggest that NMDA receptor‐mediated excitoprotection in cerebellar granule neurons is mediated via both a transcriptionally directed and a developmentally regulated postreceptor mechanism(s).

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.

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.

Inorganic Pi Increases Neuronal Survival in the Acute Early Phase Following Excitotoxic/Oxidative Insults

Abstract: Inorganic phosphate (Pi) plays a vital role in intracellular energy metabolism. Its many effects include stimulation of glucose use, enhancement of high‐energy phosphate concentrations, and modulation of cytosolic free [Ca2+]. Cultured fetal rat cortical neurons constitutively import Pi, and cytosolic levels positively correlate with [ATP], [NADPH], and energy charge. In the present study, we demonstrate that the concentration of intracellular Pi is an important determinant of acute neuronal survival after an excitotoxic or oxidative insult to cultured fetal rat cortical neurons. Extracellular Pi dose‐dependently enhanced survival of cortical neurons after exposure to NMDA at early (≤6 h) time points after termination of the insult. Pi similarly increased neuronal survival after exposure to kainic acid or H2O2. Pi‐exposed neurons had higher basal intracellular [Pi], [ATP], and [GSH], and slightly lower cytosolic free [Ca2+], compared with Pi‐deprived neurons. Pi‐exposed neurons maintained increased [ATP] after exposure to NMDA and displayed reduced formation of reactive oxygen species after exposure to kainic acid or H2O2, compared with Pi‐deprived neurons. These findings demonstrate that changes in extracellular and intracellular Pi can affect neuronal survival after excitotoxic or oxidative insults.

Methods to Facilitate Early Exploratory Testing of Novel Psychopharmacologic Agents in Humans

The starting point for this chapter follows from three assumptions: (1) substantial numbers of patients seriously ill with psychiatric illnesses such as schizophrenia and depression show inadequate therapeutic responses to all available classes of drugs; (2) decisions to take new potential psychotropic compounds into humans are more and more based on judgments as to the likelihood of there being a reasonable market for that specific compound; and (3) pharmacologic guidance can greatly enhance the efficiency of the clinical development process both in terms of reducing wasted effort in Phase I testing and of deriving maximum information concerning the appropriate dose to test the therapeutic potential of a novel compound. With regard to this latter point, it is well known that even marketed psychotropic drugs have had very misleading dose recommendations because what is required for marketing is to establish safe doses which on average have a positive effect rather than doses which produce the maximum possible benefit.