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M C Michel

Publications and source records attributed to M C Michel.

At least 163 records · Page 9Linked to original sources
PubMed1994

Functional studies on alpha 1-adrenoceptor subtypes mediating inotropic effects in rat right ventricle.

1. We have studied the alpha 1-adrenoceptor subtypes mediating inotropic effects of adrenaline in rat right ventricle and the Ca2+ sources used to elicit these effects. alpha 1A-Adrenoceptor-mediated contractile effects in rat vas deferens were studied for comparison in some cases. 2. Treatment with chloroethylclonidine did not affect the maximal beta-adrenoceptor-mediated inotropic effects in rat right ventricle or the maximal alpha 1A-adrenoceptor-mediated contractile effects in rat vas deferens; it did not alter the potency of isoprenaline in the ventricle and reduced the potency of the alpha-adrenoceptor antagonists in vas deferens only slightly. Treatment of right ventricular strips with CdCl2 markedly reduced resting tension and enhanced maximal inotropic effects of isoprenaline but did not affect its potency. 3. Inactivation of cardiac alpha 1B-adrenoceptors by treatment with chloroethylclonidine slightly enhanced the maximal inotropic effects of the full agonist, adrenaline and of several partial agonists. 4. Schild analysis of inhibition experiments with the alpha 1A-adrenoceptor-selective antagonists, 5-methyl-urapidil and (+/-)-tamsulosin, demonstrated that adrenaline causes its inotropic effects mainly via the alpha 1B-adrenoceptor subtype. Schild analysis of 5-methyl-urapidil inhibition experiments in chloroethylclonidine-treated ventricles indicated that only alpha 1A-adrenoceptors mediate the inotropic effects of adrenaline following inactivation of the alpha 1B-adrenoceptors. 5. In control ventricles the organic Ca2+ entry blocker, nitrendipine and treatment with the inorganic Ca2+ entry blocker, CdCl2 did not reduce inotropic effects of adrenaline whereas ryanodine treatment inhibited them. In contrast, nitrendipine and CdCl2 treatment had major inhibitory effects in chloroethylclonidine-treated but lacked inhibitory effects in phenoxybenzamine-treated ventricular strips. 6. We conclude that inotropic effects of adrenaline in rat heart are mediated mainly by alpha 1B-adrenoceptors via release of Ca2+ from an intracellular pool. Following inactivation of alpha 1B-adrenoceptors by chloroethylclonidine treatment, alpha lA-adrenoceptors can fully compensate and mediate inotropic effects by promoting influx of extracellular Ca2+ at least partly via voltage-operated channels.Therefore, we speculate that alpha 1B-adrenoceptors exert a tonic inhibitory effect on alpha 1A-adrenoceptors.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1994

Sensitization by dexamethasone of lymphocyte cyclic AMP formation: evidence for increased function of the adenylyl cyclase catalyst.

1. Glucocorticoids and elevations of intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) may affect lymphocyte activation, proliferation and effector functions in similar ways. Therefore, we have investigated the effects of the glucocorticoid, dexamethasone, on human lymphocyte cyclic AMP formation. 2. Treatment of resting human lymphocytes with the glucocorticoid, dexamethasone, sensitized prostaglandin E2-stimulated cyclic AMP accumulation in a time- and concentration-dependent manner. 3. In membranes of lymphocytes treated for 24 h with 100 nM dexamethasone, maximal adenylyl cyclase activity stimulated by prostaglandin E2, isoprenaline, guanosine 5'-triphosphate (GTP), forskolin and MnCl2 was significantly enhanced; the EC50 for these agents was not significantly altered. 4. beta 2-Adrenoceptor density, immunodetectable alpha-subunits of the G-proteins Gs and Gi, and pertussis toxin-substrates were not significantly altered by dexamethasone treatment. 5. In dexamethasone-treated lymphocytes, prostaglandin E2-mediated inhibition of concanavalin A-induced Ca2+ elevations was doubled compared to control cells. 6. Based on these data and the observation that enhancement of forskolin- and MnCl2-stimulated adenylyl cyclase activity could quantitatively account for the enhancement of prostaglandin E2-, isoprenaline- or GTP-stimulated adenylyl cyclase activity, we conclude that dexamethasone treatment sensitizes cyclic AMP formation in resting human lymphocytes by altering the adenylyl cyclase catalyst rather than G-proteins or hormone receptors. This results in an enhanced capability of cyclic AMP generating agonists to inhibit early steps of lymphocyte activation.

Adenylyl Cyclases↗
PubMed1994

Ontogenesis of sympathetic responsiveness in spontaneously hypertensive rats. II. Renal G proteins in male and female rats.

Previously we have reported an increased renal alpha 1- and beta-adrenergic receptor expression in male spontaneously hypertensive rats that occurred ontogenetically in parallel with blood pressure elevation. However, increased receptor numbers were not accompanied by enhanced stimulation of inositol phosphate and cyclic AMP formation, respectively, indicating relative desensitization. We have now quantified alpha-subunits of the G proteins Gs (Gs short and Gs long), G(i), and Gq by immunoblotting and pertussis toxin-catalyzed ADP-ribosylation in renal membranes from 3-, 6-, 8-, and 28-week-old normotensive and spontaneously hypertensive male Wistar-Kyoto rats; additionally, 28-week-old female normotensive and spontaneously hypertensive rats were studied. During ontogenesis of male normotensive rats, Gs short increased, Gs long remained unchanged, and G(i) alpha and Gq alpha decreased. In adult normotensive rats no sex differences were detected for Gs short, Gs long, and G(i) alpha. When male rats from the normotensive and spontaneously hypertensive strains were compared, all G protein alpha-subunits were similar in the prehypertensive phase (3 weeks). In established hypertension (28 weeks), Gs long and Gq alpha were reduced, whereas Gs short and G(i) alpha remained unchanged. Gs long was also reduced during the development of hypertension (6 and 8 weeks), whereas Gs short and G(i) alpha were not consistently altered in this phase. The reduction in Gs long seen in male adult hypertensive rats was not detectable in female hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The alpha 1A-adrenergic receptor subtype mediates biochemical, molecular, and morphologic features of cultured myocardial cell hypertrophy.

alpha 1-Adrenergic agonists induce a hypertrophic phenotype in cultured neonatal rat ventricular myocytes. Quantifiable markers of this phenotype include stimulation of phosphoinositide hydrolysis, transcriptional induction of atrial natriuretic factor (ANF) gene expression, and an increase in myocardial cell size. The aim of the present work was to determine which alpha 1-adrenergic receptor subtype mediates the acquisition of these parameters of myocardial cell hypertrophy. Phosphoinositide hydrolysis is inhibited by low concentrations of 5-methylurapidil (log Ki = -8.7) and (+)-niguldipine (log Ki = -10.6). The alpha-adrenergic receptor-induced increase in transcriptional activation of an ANF luciferase reporter gene is inhibited over the same range of concentrations of 5-methylurapidil (log Ki = -8.2) and (+)-niguldipine (log Ki = -11.2) that inhibit phosphoinositide hydrolysis. In addition, the increase in cell size that accompanies alpha-adrenergic receptor stimulation of cultured ventricular myocytes is blocked by similar concentrations of 5-methylurapidil (log Ki = -8.0) and (+)-niguldipine (log Ki = -10.6). In contrast, treatment with the alpha 1B selective antagonist chlorethylclonidine at a concentration of 10 microM had no effect on the adrenergically mediated induction of ANF luciferase reporter gene expression or the adrenergically induced increase in myocardial cell size. These findings demonstrate that pharmacologically identifiable alpha 1A-adrenergic receptors mediate not only the early effects of alpha 1-adrenergic stimulation such as phosphoinositide hydrolysis, but that they activate the signaling pathways that control transcriptional induction of the ANF luciferase reporter gene and an increase in myocardial cell size. Studies using alpha 1-adrenergic receptor cDNAs to delineate and alter the direct interaction of this receptor subtype with proximal signaling molecules, e.g. GTP binding proteins, should provide a powerful means of assessing their role in the induction of the molecular and morphologic parameters of myocardial cell hypertrophy.

Animals↗
PubMed1993

Tissue- and subunit-specific regulation of G-protein expression by hypo- and hyperthyroidism.

Thyroid hormone status has profound effects on signal transduction in various tissues throughout the body. Therefore, we quantified the signal transducing G-proteins in the rat heart, cerebral cortex, vas deferens and liver by immunoblotting and pertussis toxin labeling in response to chemically induced hypothyroidism (treatment with propylthiouracil) and hyperthyroidism (treatment with triiodothyronine). Levels of the pertussis toxin (PTX) substrates Gi alpha and Go alpha in the heart and vas deferens were inversely correlated with thyroid hormone levels, i.e. Gi alpha and Go alpha were decreased or unchanged in hyperthyroid rats and increased in hypothyroid rats compared to control animals. The cerebral cortex and liver expression of PTX substrates Gi alpha and Go alpha was not affected by changes in thyroid hormone. Regulation of Gs alpha protein was more complex in that Gs alpha was unaffected in the other tissues tested. Expression of G-protein beta-subunits was not affected by thyroid status in the heart, liver, or cerebral cortex. Our results suggest that tissue- and G-protein-specific factors are involved in the regulation of G-protein subunits by thyroid hormone. Moreover, cardiac expression of Gs alpha is upregulated by increases or decreases in the normal level of thyroid hormone.

Animals↗
PubMed1993

Prolonged isoproterenol treatment alters immunoregulatory cell traffic and function in the rat.

The mechanisms by which the sympathetic nervous system modulates functional parameters of the immune system have not been fully defined. In vivo, acute and chronic beta 2-adrenergic stimulation have been shown to have dramatic but opposing effects on circulating lymphocyte number and subset distribution in humans. In vitro studies have suggested impairment of numerous lymphocyte effector functions in the presence of catecholamines. To better define the effects of short and long term beta-adrenergic stimulation on lymphocyte migration patterns between circulating and splenic pools, as well as function, we infused rats with either low or high doses of isoproterenol over various time intervals. Specifically, we determined the number and subset composition of peripheral blood and splenic lymphocytes in addition to assessing responsivity to the T cell mitogen concanavalin A and antibody production. As a measure of in vivo lymphocyte proliferation, we also monitored the effect of these infusions on the number of peripheral blood lymphocytes and splenocytes and the splenic weight. Isoproterenol led to dose-dependent, short-lived decrements in mitogen-induced lymphocyte proliferation with concomitant decreases in circulating and splenic lymphocyte number in a subset-specific manner. Analysis of the distribution of circulating and splenic subtypes with isoproterenol treatment suggests definite but limited influences on beta-adrenergic stimulation on immunoregulatory cell traffic. We conclude that beta-adrenergic stimulation in the rat model leads to a dose-dependent, transient effect on lymphocyte proliferation and migration patterns. The relative lack of functional beta-receptors on rat lymphocytes and the propensity for early sustained receptor desensitization on exposure to agonist may account for qualitative differences seen between rats and humans.

Animals↗
PubMed1993

Comparison of the positive inotropic effects of serotonin, histamine, angiotensin II, endothelin and isoprenaline in the isolated human right atrium.

The receptor systems through which serotonin (5-HT), histamine, angiotensin II and endothelin increase the force of contraction were studied in isolated right atria from patients without apparent heart failure. All agonists increased the atrial force of contraction in a concentration-dependent manner; maximal effects, however, were significantly less than those evoked by isoprenaline or Ca2+. 5-HT and histamine, but not angiotensin II and endothelin, activated adenylate cyclase, whereas endothelin and angiotensin II stimulated inositol phosphate generation. Experiments with subtype-selective antagonists revealed that histamine effects were mediated by H2-receptors (sensitive to ranitidine), 5-HT-effects by 5-HT4-receptors (sensitive to SDZ 205-557) and angiotensin II effects by AT1-receptors (sensitive to losartan). We conclude that in human right atria the force of contraction can be increased by cyclic AMP-dependent (histamine, 5-HT) and -independent (angiotensin II, endothelin) pathways. Compared to beta-adrenoceptors, however, all other receptor systems increase the force of contraction only submaximally indicating that the beta-adrenoceptor pathway is the most important physiological mechanism to regulate force of contraction and/or heart rate in the human heart.

Adenylyl Cyclases↗
PubMed1993

Alpha 1A and alpha 1B-adrenoceptors enhance inositol phosphate generation in rat renal cortex.

We have studied the role of alpha 1A- and alpha 1B-adrenoceptors in noradrenaline- and methoxamine-stimulated inositol phosphate accumulation in rat renal cortical slices. [3H]Prazosin binding studies with and without inactivation of alpha 1B-adrenoceptors by chloroethylclonidine treatment suggested that noradrenaline lacks relevant selectivity for alpha 1-adrenoceptor subtypes. Both agonists stimulated [3H]inositol phosphate accumulation with similar maximal effects. The alpha 1A-selective antagonists 5-methyl-urapidil and (+)-niguldipine inhibited inositol phosphate formation by both agonists with shallow biphasic curves but the high affinity component was only 15%-31% and 38%-41%, respectively. The irreversible alpha 1B-selective antagonist chloroethylclonidine inhibited inositol phosphate generation by both agonists by 54%-57%. In contrast to our previous data in rat cerebral cortical slices, we conclude that in rat renal cortex both alpha 1A- and alpha 1B-adrenoceptors are involved in noradrenaline- and methoxamine-stimulated inositol phosphate generation.

Adrenergic alpha-Antagonists↗
PubMed1993

Alpha 1-adrenoceptor subtype affinities of drugs for the treatment of prostatic hypertrophy. Evidence for heterogeneity of chloroethylclonidine-resistant rat renal alpha 1-adrenoceptor.

We have used radioligand binding and inositol phosphate accumulation studies to determine the affinity at mixed alpha 1A- and alpha 1B-adrenoceptors (rat cerebral cortex and kidney), alpha 1A-adrenoceptors (rat cerebral cortex and kidney following inactivation of alpha 1B-adrenoceptors by chloroethylclonidine treatment) and alpha 1B-adrenoceptors (rat spleen) for drugs currently under investigation for the treatment of benign prostatic hypertrophy, alfuzosin, naftopidil and (-)- and (+)-tamsulosin. Alfuzosin and naftopidil had similar affinities in all model systems (approximately 10 nM and 130 nM, respectively) and lacked relevant selectivity for alpha 1-adrenoceptor subtypes. Their potency to inhibit noradrenaline-stimulated inositol phosphate formation in cerebral cortex matched their affinities as determined in the binding studies. Tamsulosin had higher affinity at alpha 1A- than at alpha 1B-adrenoceptors, and was slightly more potent than alfuzosin and naftopidil at alpha 1B- and considerably more potent at alpha 1A-adrenoceptors. However, the interaction of the tamsulosin isomers with chloroethylclonidine-insensitive (alpha 1A-like) adrenoceptors was complex. A detailed analysis of the tamsulosin data and those obtained with other drugs, most notably noradrenaline and oxymetazoline, suggested that chloroethylclonidine-insensitive alpha 1-adrenoceptors may be heterogeneous and that this heterogeneity may differ between cerebral cortex and kidney of the rat.

Alkylating Agents↗
PubMed1993

Lack of desensitization of alpha- and beta-adrenoceptor function during chronic treatment of healthy volunteers with ibopamine, an orally active dopamine receptor agonist.

In 18 healthy volunteers, in a double-blind placebo-controlled study, we investigated of whether 14 days treatment with a therapeutic dose of ibopamine (3 x 100 mg/day p.o.), respectively its active metabolite epinine, would desensitize lymphocyte beta 2- or platelet alpha 2-adrenoceptors, or alpha 1- and beta-adrenoceptor mediated (phenylephrine- and isoprenaline infusions, respectively), changes in systolic and diastolic blood pressure and heart rate. Ibopamine-treatment, which resulted in peak plasma epinine concentrations of 4-5 nmol.l-1, neither affected resting heart rate or blood pressure, nor any of the alpha- or beta-adrenoceptor parameters measured. Since in man in general long-term administration of alpha- and beta-adrenoceptor agonists desensitizes alpha- and beta-adrenoceptors, the lack of any alpha- and beta-adrenoceptor desensitizing effect of ibopamine suggests that, in the dose employed (3 x 100 mg per day), ibopamine does not exert alpha- or beta-adrenoceptor agonistic effect in humans.

Adult↗
PubMed1993

Is PP56 (D-myo-inositol-1,2,6-trisphosphate) an antagonist at neuropeptide Y receptors?

PP56 (D-myo-inositol-1,2,6,-trisphosphate) has been reported to specifically inhibit neuropeptide Y-mediated effects in vasculature, heart and brain; because of its reversible but non-competitive antagonism interaction with neuropeptide Y receptor signalling or allosteric modulation of neuropeptide Y binding have been postulated. These possibilities were tested in the present study. PP56 did not affect [125I]neuropeptide Y binding to HEL- or SK-N-MC-cells or to porcine splenic membranes. PP56 did not inhibit neuropeptide Y-stimulated Ca2+ increases in HEL- or SK-N-MC-cells or in cultured porcine aortic vascular smooth muscle cells but if anything slightly enhanced it. PP56 did not antagonize the neuropeptide Y-mediated inhibition of forskolin-stimulated cAMP accumulation in HEL-cells. We conclude that previously reported antagonistic effects of PP56 occur distal to the neuropeptide Y receptor or its second messenger systems Ca2+ and cAMP or may be restricted to neuropeptide Y receptors in certain model systems.

Animals↗
PubMed1993

Are cardiac G-proteins altered in rat models of hypertension?

OBJECTIVE: To determine whether alterations in cardiac G-protein number or function, or both, are involved in the desensitization of adenylate cyclase responsiveness in the hypertensive state. DESIGN: Quantitation of G-protein subunits in myocardial membranes from four different rat models of hypertension in comparison with respective normotensive rats. METHODS: We compared male and female adult spontaneously hypertensive rats (SHR) with age- and sex-matched Wistar-Kyoto (WKY) rats, rats from the highest-pressure quartile from an F2 generation of WKY x SHR hybrids with those from the lowest-pressure quartile, and one-kidney, one clip renal hypertensive with sham-operated rats. The function of Gs alpha was quantitated by reconstitution of cardiac cholate extracts into cyc- cell membranes with subsequent measurement of NaF-stimulated adenylate cyclase activity. The amounts of immunodetectable Gs alpha, Gi alpha, Gq alpha and G beta were determined from quantitative Western blotting experiments with [125I]-protein A detection. RESULTS: None of the parameters investigated differed significantly between hypertensive and normotensive rats in any of the models investigated. CONCLUSION: We conclude that major quantitative alterations in cardiac Gs, Gi or Gq are not a general feature of the hypertensive state.

Adenylyl Cyclases↗
PubMed1993

Norepinephrine and neuropeptide Y increase intracellular Ca2+ in cultured porcine aortic smooth muscle cells.

We used the fluorescent Ca2+ indicator Fura-2 in cultured porcine aortic smooth muscle cells (PASMC) to study effects of the sympathetic neurotransmitters norepinephrine (NE) and neuropeptide Y (NPY) on free intracellular Ca2+ (Cai). Both transmitters transiently increased intracellular Ca2+ in a concentration-dependent manner. Selective agonists and antagonists demonstrated that the NE-stimulated Cai increase is predominantly (if not exclusively) mediated by alpha 2-adrenoceptors, whereas the NPY response appears to be mediated by the peptide YY-insensitive Y3-like receptor subtype. Pretreatment of cells with pertussis toxin abolished NPY and alpha-adrenoceptor agonist-stimulated intracellular Ca2+ elevations (but not those stimulated by angiotensin II) suggesting involvement of a Gi-like G-protein. alpha 2-Adrenoceptor-stimulated Ca2+ increases resulted from mobilization from intracellular stores, whereas Y3-like NPY receptors mobilized Ca2+ from intracellular stores and also promoted Ca2+ influx.

Adrenergic alpha-Agonists↗
PubMed1993

Ontogenesis of sympathetic responsiveness in spontaneously hypertensive rats. I. Renal alpha 1-, alpha 2-, and beta-adrenergic receptors and their signaling.

We studied the ontogenetic development of renal alpha 1-, alpha 2-, and beta-adrenergic receptors and their coupling to inositol phosphate and cyclic AMP formation in spontaneously hypertensive and normotensive Wistar-Kyoto rats. alpha 1-, alpha 2-, and beta-Adrenergic receptor number was significantly increased in hypertensive compared with normotensive rats, but the increase did not precede blood pressure elevation. Despite increased alpha 1-adrenergic receptors, basal and norepinephrine-stimulated inositol phosphate formation remained unchanged in all age groups. Rat kidney contains alpha 1A- and alpha 1B-adrenergic receptors coupling to inositol phosphate formation by different mechanisms, but the relative contribution of alpha 1A- and alpha 1B-adrenergic receptors to norepinephrine-stimulated inositol phosphate formation was similar in normotensive and hypertensive rats. Despite increased beta-adrenergic receptors, basal, isoproterenol-, and forskolin-stimulated cyclic AMP accumulation was similar in normotensive and hypertensive rats. We conclude that the number but not the functional responsiveness of renal adrenergic receptors increases in spontaneously hypertensive rats. Thus, the additional receptors are unlikely to contribute to the pathophysiology of elevated blood pressure in this model.

Animals↗
PubMed1993

Effects of ketotifen on human lymphocytes in vitro and in vivo.

The effects of the antiasthmatic drug ketotifen (CAS 34580-13-7) on human mononuclear leukocytes were studied in vitro and in vivo. In vitro ketotifen concentration-dependently inhibited mitogen-stimulated lymphocyte proliferation. High ketotifen concentrations also inhibited T-lymphocyte mitogen- and adenosine triphosphate stimulated increases in intracellular Ca2+ in lymphocytes and the U937 human monocyte precursor cell line, respectively; this involved inhibition of both Ca2+ influx and intracellular mobilization. In in vivo experiments, treatment of healthy volunteers with 1 mg ketotifen b.i.d. for 7 d did not alter the number or subset composition of circulating lymphocytes. Moreover, the mitogen-stimulated in vitro proliferation of lymphocytes obtained before and after ketotifen treatment in vivo was similar. It is concluded that high ketotifen concentrations can inhibit the activation of resting lymphocytes in vitro but standard ketotifen treatment does not notably affect the number of function of circulating lymphocytes in vivo.

Adenosine Triphosphate↗
PubMed1993

Selective irreversible binding of chloroethylclonidine at alpha 1- and alpha 2-adrenoceptor subtypes.

We have determined the alkylating effects and affinity of chloroethylclonidine at alpha 1- and alpha 2-adrenoceptor subtypes in saturation and competition radioligand binding studies. Treatment with chloroethylclonidine (10 microM, for 30 min at 37 degrees, with subsequent washout) abolished [3H]prazosin binding to alpha 1B-adrenoceptors in rat spleen almost completely and reduced specific binding in rat kidney and cerebral cortex by a percentage comparable to the known alpha 1B-adrenoceptor content of these tissues. Chloroethylclonidine treatment also markedly reduced [3H]rauwolscine binding to human platelet and kidney membranes but did not affect [3H]rauwolscine binding to rat kidney. Similar chloroethylclonidine treatment (10 microM, 20 min at 37 degrees) reduced the number of detectable alpha 2-adrenoceptors in cell lines transfected with the alpha 2-C10 or alpha 2-C4 gene but not in those transfected with alpha 2-C2 adrenoceptors. In concentration-response experiments, higher chloroethylclonidine concentrations were required for inactivation of human platelet alpha 2A-adrenoceptors, compared with rat spleen alpha 1B-adrenoceptors, and a smaller maximal inactivation was achieved. The lack of inactivation of rat alpha 1A- and alpha 2B- and human alpha 2-C2-adrenoceptors was not due to a lack of chloroethylclonidine binding, because the affinity of chloroethylclonidine at these subtypes, as determined in competition binding experiments, was at least as high as the apparent affinity at the alkylated subtypes. alpha 2A-Adrenoceptor alkylation by chloroethylclonidine treatment was functionally relevant, because it significantly reduced alpha 2A-adrenoceptor-mediated Ca2+ elevations in HEL cells. We conclude that chloroethylclonidine binds to all major alpha-adrenoceptor subtypes and irreversibly inactivates not only alpha 1B-adrenoceptors but also alpha 2A- and alpha 2C-adrenoceptors, whereas alpha 1A- and alpha 2B-adrenoceptors are relatively resistant to its alkylating action, although they can bind chloroethylclonidine.

3T3 Cells↗
PubMed1992

Dissociation between phytohaemagglutinin-stimulated generation of inositol phosphates and Ca2+ increase in human mononuclear leucocytes.

We have tested whether phytohaemagglutinin (PHA)-stimulated generation of inositol phosphates (IP) and increases in intracellular Ca2+ can be dissociated in human mononuclear leucocytes. Lowering the incubation temperature from 37 degrees to 25 degrees C decreased PHA-stimulated IP generation by more than 80%, but only marginally affected PHA-stimulated Ca2+ increases. In the absence of extracellular Ca2+, PHA did not stimulate IP generation or Ca2+ increases, although PHA binding to its acceptor sites was not impaired. Increasing extracellular Ca2+ up to 0.15 mM enhanced PHA-stimulated PHA generation but this increase was attenuated by further increasing extracellular Ca2+ to 2.6 mM. Increasing extracellular Ca2+ to 0.3 mM also enhanced PHA-stimulated Ca2+ increases, and further increasing extracellular Ca2+ did not affect it. Co-treatment with 100 microM-prostaglandin E2 completely abolished PHA-stimulated IP generation, but inhibited Ca2+ increases by only 20-30%. These results could be explained by IP-generation-independent Ca2+ increases or by non-linear coupling of IP generation to Ca2+ increases. Since the PHA concentrations required to increase Ca2+ were greater than those required for IP generation, the latter hypothesis can be excluded. Furthermore, the Ca2+ ionophore ionomycin increased intracellular Ca2+ and weakly stimulated IP generation, but with very similar concentration-response relationships. Our data suggest that PHA-stimulated IP generation and Ca2+ increases in human mononuclear leucocytes mainly occur independently of one another rather than sequentially.

Calcium↗