Purinergic receptors in the nucleus tractus solitarius mediate distinct cardiorespiratory response patterns

The purpose of the study was to examine the role of brainstem purinergic receptors in mechanisms of cardiorespiratory control mediated by the nucleus tractus solitarius (NTS), the major integrative site in the brainstem involved in the regulation of cardiorespiratory function and the reflex coordination of cardiorespiratory response patterns. Microinjections of selective agonists for purinergic receptor subtypes were made into the caudal NTS of anesthetized, spontaneous breathing rats. CGS 21680, a selective agonist for adenosine A_2a receptors, elicited pronounced, dose-related decreases in blood pressure, whereas cyclopentyladenosine (CPA), a selective agonist at adenosine A₁ receptors, elicited dose-related increases in blood pressure. These depressor and pressor response patterns were completely and selectively blocked, respectively, by pretreatment with CGS 15943a, a selective A_2a receptor antagonist, and by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) a selective A₁ receptor antagonist. 5′-N-ethylcarboxamidoadenosine (NECA), an analog exhibiting mixed agonist actions at adenosine receptor subtypes, elicited both depressor and pressor responses depending on the dosage. Additionally, in contrast to the findings with CGS 21680 and CPA, only NECA elicited very pronounced, dose-related decreases in respiratory rate at dosages which did not affect blood pressure. Related lines of evidence suggested that NECA-induced depression of respiration was mediated via A_2b receptors. Microinjections of α,β-MeATP, a selective agonist for P_2x receptors, elicited apnea, and these effects were blocked by suramin, a selective antagonist at P_2x receptors, but not by DMPX, a methylxanthine exhibiting mixed antagonist properties at P₁ adenosine receptors. Moreover, similar microinjections of 2-MeSATP, a selective agonist at P_2x receptors, were ineffective in eliciting cardiorespiratory response patterns and it is likely that brainstem purinergic receptors may therefore play a crucial role under both physiologic and pathophysiologic conditions. © 1993 Wiley-Liss, Inc.     

Voltage and ionic regulation of human serotonin transporter in Xenopus oocytes

1. The serotoninergic system is known to be involved in the control of multiple behavioural and physiological functions. The serotonin (5-hydroxtryptamine; 5-HT) transporter (SERT), which controls the synaptic 5-HT concentration through re-uptake of this neurotransmitter into presynaptic terminals, has been a primary therapeutic target for various psychiatric and peripheral disorders. The aim of the present study was to identify the regulatory mechanism(s) of the human SERT (hSERT) in heterologously expressed oocytes. 2. The hSERT cRNA was transcribed in vitro and injected into Xenopus oocytes. The 5-HT-induced transporter currents were measured by voltage clamp. The effects of extracellular sodium or chloride were studied by replacement perfusion with tetramethylammonium-chloride (96 mmol/L) or sodium acetate (96 mmol/L). In addition, to alter the internal calcium concentration, CaCl2 (50 micromol/L) and inositol triphosphate (IP3; 50 micromol/L), with or without EGTA (2.5 mmol/L), were injected into oocytes. The specificity of 5-HT-sensitive currents was determined by the use of the SERT antagonist desipramine and niflumic acid to block background chloride currents. 3. The hSERT-expressing oocytes displayed voltage-dependent, 5-HT-induced currents that increased at negative potentials. Replacing extracellular sodium or chloride significantly decreased the hSERT currents by 89 and 45%, respectively (P < 0.05, n = 7 each). Injection of IP3 or CaCl2 increased the hSERT currents by approximately 65% (P < 0.05; n = 10 each) and the effect of IP3 was abolished by preinjection of EGTA. 4. These results demonstrate that hSERT activity is not only voltage dependent, but is also affected by intracellular calcium and extracellular sodium and chloride.     

Inositol derivatives modulate spontaneous transmitter release at the frog neuromuscular junction

One of the consequences of G-protein-coupled receptor activation is stimulation of phosphoinositol metabolism, leading to the generation of IP3 and its metabolites 1,3,4,5-tetrakisphosphate (IP4) and inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Previous reports indicate that high inositol polyphosphates (IP4 and IP6) are involved in clathrin-coated vesicular recycling. In this study, we examined the effects of IP4 and IP6 on spontaneous transmitter release in the form of miniature endplate potentials (MEPP) and on enhanced vesicular recycling by high K+ at frog motor nerve endings. In resting conditions, IP4 and IP6 delivered intracellularly via liposomes, caused concentration-dependent increases in MEPP frequency and amplitude. Pretreatment with the protein kinase A (PKA) inhibitor H-89 or KT 5720 reduced the IP4-mediated MEPP frequency increase by 60% and abolished the IP6-mediated MEPP frequency increases as well as the enhancement in MEPP amplitude. Pretreatment with antibodies against phosphatidylinositol 3-kinase (PI 3-K), enzyme also associated with clathrin-coated vesicular recycling, did not alter the IP4 and IP6-mediated MEPP frequency increases, but reduced the MEPP amplitude increase by 50%. In our previous reports, IP3, but not other second messengers releasing Ca2+ from internal Ca2+ stores, is able to enhance the MEPP amplitude. In order to dissociate the effect of Ca2+ release vs. metabolism to IP4 and IP6, we evaluated the effects of 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate (3F-IP3), which is not converted to IP4 or IP6. 3F-IP3 produced an increase then decrease in MEPP frequency and a decrease in MEPP amplitude. In elevated vesicle recycling induced by high K+-Ringer solution, IP4 and IP6 have similar effects, except decreasing MEPP frequency at a higher concentration (10(-4) M). We conclude that (1) high inositol polyphosphates may represent a link between IP3 and cAMP pathways; (2) the IP3-induced increase of MEPP amplitude is likely to be due to its high inositol metabolites; (3) PI 3-K is not involved in the IP4 and IP6-mediated MEPP frequency increases, but may be involved in MEPP size.     

Existence and alpha 1-adrenergic stimulation of inositol polyphosphates in mammalian heart.

The concentration-response curves and the time course of the effects of phenylephrine (0.01-100 microM) on force of contraction and on inositol polyphosphates in isolated electrically stimulated perfused rat hearts (Langendorff technique) were studied. A nonradiometric high performance liquid chromatography metal dye detection technique was used to determine absolute concentration masses/changes of inositol polyphosphates in heart. Products measured after separation with high performance liquid chromatography were inositol 1,4,5-trisphosphate (1,4,5-IP3), inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4) and its isomer 1,3,4,6-IP4, inositol 1,3,4,5,6-pentakisphosphate (1,3,4,5,6-IP5), and inositol hexakisphosphate (IP6). 1,4,5-IP3 (significant at 10 microM) and both IP4 isomers (significant at 1 microM) increased after alpha-adrenoceptor stimulation, whereas 1,3,4,5,6-IP5 and IP6 remained unaffected. Phenylephrine had a concentration-dependent positive inotropic effect (significant at 1 microM). All effects were antagonized by the alpha 1-adrenoceptor antagonist prazosin (0.1 microM), indicating receptor-mediated effects. In a time course study 1,4,5-IP3 was the first compound to increase significantly, within 1 min after stimulation; this rise was followed by an increase in 1,3,4,5-IP4 beginning within 2 min. The increase in all other inositol polyphosphates was slower (5-10 min). The increase in the force of contraction started at 2 min. For comparison, the effects of the beta-adrenoceptor agonist isoprenaline were studied. Isoprenaline produced a positive inotropic effect similar to that of phenylephrine, but all inositol polyphosphates remained unaffected. In conclusion, for the first time the existence of 1,3,4,5,6-IP5 and IP6 was observed in the heart. However, the physiological role of these inositol polyphosphate isomers in the heart remains to be elucidated, because, from the time course, they appear to have no acute intracellular second messenger function. Increased inositol polyphosphate turnover may be involved in the mechanism(s) whereby alpha 1-adrenoceptor stimulation produces an increase in myocardial force of contraction. Because the increase in 1,4,5-IP3 precedes and that in 1,3,4,5-IP4 coincides with the increase in the force of contraction, 1,4,5-IP3 may initiate and 1,3,4,5-IP4 may maintain the positive inotropic effect of alpha 1-adrenoceptor agonists.     

Selective cardiorespiratory effects mediated by mu opioid receptors in the nucleus ambiguus

The respiratory and cardiovascular effects of the highly selective mu opioid agonist, D-Ala2, MePhe4, Gly- ol5 enkephalin ( DAGO ) and the relatively selective delta agonist, D-Leu5 enkephalin (DADL) were compared following injection (0.1 microliter) into the nucleus ambiguus (NA) of spontaneously-breathing and artificially-respired, pentobarbital-anesthetized rats. In non-ventilated animals, the opioids elicited dose-related (3 X 10(-11) -3 X 10(-9) M), naloxone-reversible depression of respiratory rate (RR) without altering the tidal volume. Mean arterial pressure (MAP) was unchanged at small doses and decreased at the largest dose; heart rate (HR) was unchanged. In artificially-respired animals, both peptides elicited dose-related, naloxone-reversible increases in mean arterial pressure and heart rate; DAGO was significantly more potent than DADL (P less than 0.01). Given the relative potency and selectivity of the opioids tested, these findings are consistent with the conclusion that mu receptors may selectively mediate the respiratory and cardiovascular actions of opioids in an important brain stem cardiorespiratory center in the rat. Moreover, these data indicate the importance of respiratory effects on the cardiovascular activity of centrally administered opioids.     

Formation of inositol phosphates in synaptoneurosomes of guinea pig brain: stimulatory effects of receptor agonists, sodium channel agents and sodium and calcium ionophores

Carbamylcholine, norepinephrine, histamine and glutamate stimulated the formation of [3H]inositol phosphates in [3H]inositol-labelled synaptoneurosomes obtained from the cortex, striatum and hippocampus of the guinea pig. Synaptoneurosomes prepared from the cerebellum did not respond to receptor agonists. Agents that enhance the influx of sodium ions through voltage-sensitive channels, such as batrachotoxin, scorpion venom and pumiliotoxin B, or a sodium ionophore, monensin, stimulated the formation of [3H]inositol phosphates in synaptoneurosomes from all four regions of the brain. Neither calcium channel blockers nor receptor antagonists reduced the responses to batrachotoxin. Ionomycin, a calcium ionophore, also stimulated the formation of [3H]inositol phosphates in synaptoneurosomes from all four regions of the brain. A phorbol ester inhibited the formation of [3H]inositol phosphates, elicited by either receptor agonists or by sodium channel agents. The major [3H]inositol-labelled lipid in the synaptoneurosomes was phosphatidylinositol as analyzed by thin layer chromatography. While the hydrolysis of phosphatidylinositol elicited by carbamylcholine resulted in an increase of lipid-labelling with [3H]inositol, sodium channel agents caused a decrease in the incorporation of [3H]inositol. The results indicate that intracellular sodium may have a regulatory role in the turnover of phosphatidylinositol, and that, unlike the receptor-mediated responses, this regulation is present in all regions of the brain.     

Endothelin stimulates phosphatidylinositol turnover in rat right and left atria

The effect of endothelin on phosphatidylinositol (PI) turnover has been investigated in isolated rat atria by measuring the generation of inositol phosphates (IPs) following [3H]inositol phospholipid labelling. In the presence of 10 mM LiCl, endothelin caused dose-dependent increases in the accumulations of inositol mono-, bis- and tris-phosphate (IP1, IP2 and IP3) which were maximal at 10(-6) M endothelin. The dose-response relationship was similar in right and left atria, but right atria showed a higher maximal IP response. Endothelin produced a rapid and transient stimulation of IP3 accumulation, which was maximal at 30 s followed by a slower increase which continued linearly past 20 min. During both the initial phase and the sustained phase the only isomer of IP3 present at detectable levels was the 1,4,5-isomer. As with endothelin, responses to noradrenaline also were higher in right atria compared with left atria and showed a biphasic pattern of release of IP3. These data demonstrate that endothelin receptors in rat atria are coupled to stimulation of the PI turnover pathway in an apparently similar manner to alpha 1-adrenoceptors. The PI pathway may be important in mediating the reported cardiac actions of endothelin.     

Adenosine reduces agonist-induced production of inositol phosphates in rat aorta

In rat aortic strips rendered permeable with digitonin, inositol trisphosphate induced an efflux of 45Ca from the tissue. This release was not affected by adenosine. In tissues not treated with digitonin the contents of inositol trisphosphate (IP3) and its metabolite inositol 1-phosphate (IP1) were significantly enhanced by noradrenaline in the lithium-treated rat aorta. Adenosine was without effect on levels of IP1 or IP3 in tissues which had not been pretreated with noradrenaline, however, the noradrenaline-enhanced tissue content of IP1 was reduced by adenosine in a dose-dependent manner. The reduction in IP1 content by adenosine was enhanced by the uptake blocker dipyridamole (10 microM) and was blocked by the adenosine receptor antagonist 8-phenyltheophylline (10 microM). Adenosine may therefore lower production of inositol phosphates and thus reduce the stimulated release of calcium from intracellular stores. It is proposed that a reduction in phosphatidylinositol turnover may play a role in adenosine-mediated relaxation of blood vessels.     

吗啡长时程作用下小鼠脑组织磷酸肌醇含量和PKC活性的变化

目的旨在进一步阐明阿片依赖中PLC-PKC信号系统的变化与作用。用小鼠吗啡依赖模型,观察脑组织磷酸肌醇含量、PKC活性变化。结果发现:纹状体IP及IP3、大脑皮质IP含量明显升高,二者磷酸肌醇总量显著增加;胞质可溶相PKC活性在大脑皮质及小脑明显升高,在纹状体显著下降。膜相PKC活性在纹状体明显升高,在小脑和海马明显降低;PKC抑制剂可抑制吗啡依赖的产生。结果提示,吗啡依赖小鼠纹状体磷酸肌醇含量的增加意味着PLC的活化,这可能与纹状体中PKC的激活有关,而该PKC的激活可能参与吗啡依赖的形成。     

Local anesthetics: comparison of effects on batrachotoxin-elicited sodium flux and phosphoinositide breakdown in guinea pig cerebral cortical synaptoneurosomes

Local anesthetics inhibited the sodium influx and the inositol phosphate accumulation elicited by the sodium channel activator batrachotoxin in guinea pig cortical synaptoneurosomes. Inhibitory effects of local anesthetics on sodium influx correlated with inhibitory effects on binding of a tritiated batrachotoxin analog to sodium channels in synaptoneurosomes. There was also a correlation between inhibitory effects on sodium influx and on inositol phosphate accumulation; most local anesthetics inhibited sodium influx at concentrations similar to those required for inhibition of inositol phosphate accumulation. Indeed, euprocin, bupivacaine, lidocaine, and certain analogs were nearly equipotent with respect to inhibition of sodium influx and inositol phosphate accumulation. Local anesthetics also inhibited inositol phosphate accumulation that was induced by carbamylcholine through both a tetrodotoxin-sensitive and tetrodotoxin-insensitive pathway. Certain local anesthetics, such as dibucaine, inhibited the tetrodotoxin-sensitive pathway with higher potency than for the tetrodotoxin-insensitive pathway, while others, such as quinacrine, inhibited tetrodotoxin-sensitive and tetrodotoxin-insensitive pathways with equal potency. Diphenhydramine and chlorpromazine appeared to inhibit carbamylcholine-elicited phosphoinositide breakdown through blockade of muscarinic cholinergic receptors rather than because of local anesthetic activity of inhibitory effects on phospholipase C.     

Specific binding of inositol hexakisphosphate (phytic acid) to adrenal chromaffin cell membranes and effects on calcium-dependent catecholamine release.

In a membrane preparation of cultured bovine adrenal chromaffin cells, [3H]inositol hexakisphosphate ([3H]InsP6) was shown to bind specifically with a Kd of 90 nM and a Bmax of 700 fmol/mg protein. The Hill coefficient was not significantly different from unity. The association of [3H]InsP6 was slow, with equilibrium binding being reached within 10 min. The dissociation of [3H]InsP6 showed monophasic kinetics. In [3H]InsP6 competition binding experiments, we found that reduction in the number of phosphorylated sites in inositol resulted in a gradual loss of binding potency. In intact bovine adrenal chromaffin cells, InsP6 elicited a concentration-dependent facilitation of 45C2+ influx along with the release of the catecholamines, epinephrine and norepinephrine. The latter responses were slower and longer-lasting than responses to depolarizing stimuli, such as nicotine and high K+. The catecholamine release required the presence of extracellular Ca2+. In good agreement with the binding studies, lower inositol phosphates displayed reduced secretagogue potency. In conclusion, in bovine adrenal chromaffin cells, InsP6 appeared to bind to specific sites and elicited Ca2+ influx and catecholamine release.     

Autonomic and respiratory responses to microinjection of ATP into the intermediate or caudal nucleus tractus solitarius in the working heart-brainstem preparation of the rat

1. Activation of peripheral chemoreceptors with KCN in the working heart-brainstem preparation from young male Wistar rats (70-90 g) increases phrenic (PNA; +105 +/- 18%) and thoracic (tSNA; +44 +/- 6%) sympathetic nerve activity compared with baseline and reduces heart rate (HR; from 377 +/- 27 to 83 +/- 6 b.p.m.). 2. Microinjections of increasing doses of ATP (1, 5, 25, 100 and 500 mmol/L; n = 7) into the intermediate nucleus tractus solitarius (NTS) produced a dose-dependent reduction in PNA (from -6 +/- 3 to -82 +/- 1%) and in HR (from -12 +/- 4 to -179 +/- 47 b.p.m.). Microinjections of ATP into the intermediate NTS also produced a reduction in tSNA (from -3 +/- 3 to -26 +/- 5%), which was not dose dependent. 3. Microinjections of ATP into the caudal NTS (n = 5) produced a dose-dependent increase in PNA (from 0.2 +/- 3 to 115 +/- 27%) and minor changes in HR and tSNA, which were not dose dependent. 4. The data show that microinjection of ATP into distinct subregions of the NTS produces different respiratory and autonomic responses and suggest that ATP in the caudal NTS is involved in the respiratory but not in the sympathoexcitatory component of the chemoreflex.     

Effects of carbachol and (-)-N6-phenylisopropyladenosine on myocardial inositol phosphate content and force of contraction.

1. The effects of carbachol and the A1-adenosine receptor agonist (-)-N6-phenylisopropyladenosine (PIA) on force of contraction and inositol lipid metabolism were studied in electrically driven left auricles and papillary muscles isolated from guinea-pig hearts. Both carbachol and PIA (0.01-10 microM) had concentration-dependent negative inotropic effects in auricles. In papillary muscles PIA had no inotropic effect. Carbachol also had no inotropic effect at low concentrations (0.01-1 microM) but at 10-100 microM it exerted a slight positive inotropic effect. 2. In auricles and papillary muscles both carbachol and PIA concentration-dependently increased inositol trisphosphate (IP3; significant at 1 microM). Accordingly phosphatidylinositol bisphosphate (PIP2), the precursor of IP3, was reduced. All effects of carbachol and PIA were antagonized by atropine (10 microM) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 20 microM) respectively, indicating receptor-mediated effects. 3. In auricles the negative inotropic effects of carbachol and PIA preceded the increase in IP3. 4. In papillary muscles the increase in IP3 preceded the slight positive inotropic effect of carbachol, indicating that the M-cholinoceptor-mediated increase in IP3 and force of contraction may be related. However, PIA showed a comparable increase in IP3 but no inotropic effect, indicating a dissociation between those parameters. 5. In conclusion, in previous studies a close relation between increases in IP3 and force of contraction has been shown after alpha 1-adrenoceptor stimulation. The present study with carbachol supports this view. However, the present data for PIA could not show such a close relationship, questioning the role of IP3 as an endogenous regulator of force of contraction.     

Meso-2,3-dimercaptosuccinic acid induces calcium transients in cultured rhesus monkey kidney cells

The maintenance of intracellular Ca2+ homeostasis is critical to many cellular functions that rely on the calcium ion as a messenger. While attempting to characterize the effects of lead on intracellular calcium levels ([Ca2+]i) in LLC-MK2 Rhesus Monkey kidney cells, we observed that treatment with the metal chelating drug, meso-2,3-dimer-captosuccinic acid (DMSA) evoked transient increases in [Ca2+]i. Changes in [Ca2+]i were monitored using the Ca2+ indicator dye Fura-2 and a dual wavelength fluorescence imaging system. In the presence of 2 mM extracellular Ca2+, DMSA treatment caused a concentration-dependent (15-500 microM) transient increase in [Ca2+]i returning to baseline levels within 30-60 s. Pharmacologic concentrations of DMSA (30 microM) stimulated a three-fold increase in [Ca2+]i, which was spatiotemporally comparable to Ca2+ transients induced by other calcium agonists. Depletion of inositol trisphosphate (IP3)-sensitive [Ca2+]i stores with the smooth endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin did not prevent DMSA-elicited increases in [Ca2+]i, suggesting that Ca2+ mobilized by DMSA was either extracellular or from an non-IP3 releasable Ca2+ pool. Treatment with glutathione, cysteine, or 2-mercaptoethanol caused similar but not identical calcium transients. Adenosine-5'-trisphosphate (ATP) also elicited transient increases in [Ca2+]i similar to those of DMSA. No transient increases in [Ca2+]i were elicited by DMSA or ATP in the absence of extracellular calcium. These data indicate that DMSA and other sulfhydryl compounds trigger an influx of extracellular calcium, suggesting a previously unobserved and unanticipated interaction between DMSA and the Ca2+ messenger system.     

Investigation of Acute Cardiovascular and Respiratory Toxicity of HLö 7 Dimethanesulfonate and HI 6 Dichloride in Anaesthetized Guinea-Pigs

The bis-pyridinium dioxime HLö 7 is considered to possess promising therapeutic properties in the treatment of organophosphate poisoning. Acute circulatory and respiratory effects of HLö 7 and HI 6 were therefore compared in anaesthetized guinea-pigs. Female Pirbright white guinea-pigs were anaesthetized with urethane and the carotid artery, jugular vein and trachea were cannulated. Saline or atropine, 10 mg/kg, or HLö 7 or HI 6 (30 or 100 μmol/kg, each) or atropine plus oxime were injected intravenously after base line measurements. Respiratory and circulatory parameters were recorded for 60 min., then blood was drawn for AChE measurement. Injection of HLö 7 or HI 6 alone resulted in a temporary, dose-dependent hypotension, an almost unchanged heart rate and a slight respiratory stimulation. A more severe hypotension appeared after the administration of atropine plus HLö 7 or HI 6. In these groups heart rate and respiration were markedly stimulated. Measurement of AChE activity in blood samples revealed no impairment by HLö 7 or HI 6 with or without atropine. These results suggest that HLö 7 has only transient effects on the cardiorespiratory system after intravenous administration and its safety regarding acute circulatory and respiratory toxicity is comparable to HI 6.     

Noradrenaline-stimulated inositol phosphate accumulation in arteries from spontaneously-hypertensive rats.

1. The effects of noradrenaline upon polyphosphoinositide (PPI) breakdown was investigated by measuring the accumulation of inositol phosphates (IPs) in tail arteries from normo- (WKY) and spontaneously-hypertensive (SHR) rats. 2. Noradrenaline (10(-7)-10(-3) M) evoked a concentration-dependent increase in total IP accumulation in both WKY and SHR rats but no significant differences between the populations were detected. 3. In contrast, significant differences in the accumulation of the individual IPs, which contributed to the total IP, occurred. A significantly greater noradrenaline-stimulated accumulation of inositol trisphosphate (IP3) was observed in tissues from SHR compared with those from WKY rats at each effective concentration of noradrenaline. This was paralleled by an equivalent reduction in inositol monophosphate (IP1) accumulation, consistent with the lack of a significant difference in noradrenaline-stimulated total IP accumulation between the two populations. 4. In time course studies, an enhanced noradrenaline-induced accumulation of IP3, in SHR compared to WKY rats, occurred from the earliest time point studied after the addition of the catecholamine both in the presence and absence of LiCL (10 mM). In the presence of LiCl (10 mM) no significant difference in noradrenaline-evoked total IP accumulation between SHR and WKY rats was observed; in the absence of LiCl noradrenaline-evoked a greater total IP accumulation in SHR than in WKY rats at all time points investigated. 5. These studies suggest that the main reason for the enhanced noradrenaline-induced accumulation of IP3 in arteries from SHR rats is a reduced rate of dephosphorylation of both IP3 and inositol bisphosphate (IP2) rather than a greater formation of IP3 from PPIs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-mediated inositol phospholipid hydrolysis in astrocytes

Astrocyte-enriched cultures of the neonatal rat cortex were incubated for 24 h with [3H]inositol to prelabel the membrane inositol phospholipids. Exposure of the cultures to either noradrenaline or carbachol in the presence of Li+ produced a time- and dose-dependent accumulation of intracellular [3H]inositol phosphates. The separation of the individual inositol phosphates formed in response to receptor stimulation revealed that the major 3H-metabolite accumulated under these conditions was inositol monophosphate but that at least some of this was due to the initial formation of inositol trisphosphate. The use of selective receptor antagonists showed that noradrenaline- and carbachol-induced [3H]inositol phosphate accumulation was the result of the activation of alpha 1-adrenoceptors and muscarinic acetylcholine (probably of the M1 subtype) receptors respectively. Agonist-evoked [3H]inositol phosphate accumulation were found to be additive but the simultaneous addition of agonists and the Ca2+ ionophore A23187, which also promoted inositol phospholipid hydrolysis, was not. Agonist-induced [3H]inositol phosphate accumulation was only partially dependent on extracellular Ca2+, whilst that elicited by A23187 was entirely Ca2+-dependent. The results suggest that alpha 1-adrenoceptors and muscarinic acetylcholine receptors in these cultures are present either on the same cells and linked to separate inositol lipid pools or associated with different subpopulations of astrocytes in these cultures. Moreover, inositol lipids other than phosphatidylinositol 4,5-bisphosphate may be hydrolysed in response to agonist stimulation.     

A beta-carboline derivative (ZK 93426) counteracts the cardiorespiratory depressant effects of intravenous midazolam.

The purpose of this study was to test the effects of the new beta-carboline ZK 93426 on midazolam-induced cardiorespiratory depression. Seven pentobarbital-anesthetized (35 mg/kg i.p.) cats were treated with intravenous midazolam (2 mg/kg) while monitoring the respiratory minute volume (VE), tidal volume, respiratory rate, blood pressure, heart rate and expired CO2. Midazolam caused significant decreases in VE (p less than 0.05) and blood pressure (p less than 0.05). ZK 93426 (5 mg/kg i.v.) antagonized these effects and produced significant increases in VE and blood pressure that resulted in the return of these variables to premidazolam control values. In 4 animals with morphine-induced respiratory depression, intravenous ZK 93426 failed to antagonize the respiratory effects of morphine. Administration of intravenous ZK 93426 alone to 4 pentobarbital-anesthetized animals also failed to produce significant changes in cardiorespiratory activity. We conclude that ZK 93426 is effective in counteracting the cardiorespiratory depressant effects of midazolam and that these effects appear to be specific. The present data suggest that this compound may be useful for the treatment of benzodiazepine oversedation and overdose.     

Leukotriene-induced hydrolysis of inositol lipids in guinea pig lung: mechanism of signal transduction for leukotriene-D4 receptors

Addition of leukotriene D4 (LTD4) to [3H]myo-inositol-labeled guinea pig lung induced rapid breakdown of inositol lipids. Formation of [3H]inositol trisphosphate was rapid, with a peak of 140-160% of the control level, 30 sec post-treatment. Formation of [3H]inositol bisphosphate and [3H]inositol monophosphate ([3H]IP1) was also rapid in the presence of LiCl. LTD4-induced [3H]IP1 formation was concentration dependent, stereoselective, and not inhibited by the cyclooxygenase inhibitor, indomethacin. Agonist analogs of LTD4 and leukotriene E4 also induced dose-dependent increases in the synthesis of [3H]IP1. The rank order potency of the agonist-induced [3H]IP1 formation was equivalent to those reported for LTD4 receptor binding, smooth muscle contraction, and thromboxane B2 biosynthesis. Furthermore, a specific receptor antagonist, SKF 102922, inhibited LTD4-induced [3H]IP1 formation in guinea pig lung. These studies suggest that LTD4 may interact with membrane receptor and activate a phospholipase C, which in turn induces the hydrolysis of inositol lipids. The hydrolysis products, diacylglycerol and inositol trisphosphate, can be regarded as the intracellular messengers for LTD4 receptors in guinea pig lung. This concept may explain a variety of pharmacological effects of leukotrienes in different types of target cells or tissues.