Evidence for the existence of inositol tetrakisphosphate in mammalian heart. Effect of alpha 1-adrenoceptor stimulation

The time course of the effects of phenylephrine (10 mumol/l) on force of contraction and on inositol phosphates in electrically driven left auricles from rat hearts labeled with [3H]inositol was studied. All experiments were performed in the presence of propranolol (1 mumol/l) and LiCl (10 mmol/l). Products measured after separation with high-performance liquid chromatography were inositol 1-phosphate (1-IP1), inositol 1,4-bisphosphate (1,4-IP2), inositol 1,3,4-trisphosphate (1,3,4,-IP3), inositol 1,4,5-trisphosphate (1,4,5-IP3), and inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4). All inositol phosphates increased after stimulation with phenylephrine. 1,4,5-IP3 was the first compound to rise maximally within 30 seconds; this rise was followed by an increase in 1,3,4,5-IP4 and 1,4-IP2 beginning within 2 minutes. The increase in 1,3,4-IP3 and 1-IP1 was slower and did not reach steady state within 15 minutes. The positive inotropic effect of phenylephrine was maximal after 5 minutes. It is concluded that the increase in the presumed second messengers 1,4,5-IP3 and 1,3,4,5-IP4 coincides with the positive inotropic effect after alpha 1-adrenoceptor stimulation. Since the increase in 1,4,5-IP3 precedes the increase in force of contraction, 1,4,5-IP3 may initiate the positive inotropic effect of alpha 1-adrenoceptor agonists and 1,3,4,5-IP4 maintains the increase in force of contraction.     

Impaired inotropic response to alpha 1- but not to beta-adrenoceptor stimulation in isolated hearts from spontaneously hypertensive rats.

OBJECTIVES: Hypertension in humans and experimental animals is known to be associated with an increase in left ventricular myocardial mass. The development of cardiac hypertrophy is not caused by increased blood pressure alone; the autonomic nervous system may also play an important role. DESIGN: The functional responses to the beta-adrenoceptor agonists isoprenaline, dobutamine, salbutamol and terbutaline, and the alpha 1-adrenoceptor agonists methoxamine, cirazoline and phenylephrine were studied in isolated (Langendorff) hearts from spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) controls. The results were compared with data from radioligand binding experiments. RESULTS: There was no significant difference in the increase of left ventricular pressure induced by all beta-adrenoceptor agonists studied in SHR and WKY rat hearts. Although there was no significant difference in the response to phenylephrine, the inotropic responses to cirazoline and methoxamine proved to be significantly weaker in hearts from SHR than in those from WKY rats. Binding experiments with 3H-prazosin revealed no differences in density or affinity for cardiac tissues from SHR and WKY rats. CONCLUSIONS: Long-standing hypertension leads to an impaired response of the isolated heart to alpha 1-adrenoceptor stimulation, without changes in alpha 1-receptor density or affinity. It seems likely that changes in postreceptor events are responsible for the impaired inotropic response to alpha 1-adrenoceptor agonists in hearts from SHR.     

The phosphatidylinositol (PI)-5-phosphate 4-kinase type II enzyme controls insulin signaling by regulating PI-3,4,5-trisphosphate degradation

Phosphatidylinositol-5-phosphate (PI-5-P) is a newly identified phosphoinositide with characteristics of a signaling lipid but no known cellular function. PI-5-P levels are controlled by the type II PI-5-P 4-kinases (PIP4K IIs), a family of kinases that converts PI-5-P into phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). The PI-5-P pathway is an alternative route for PI-4,5-P2 synthesis as the bulk of this lipid is generated by the canonical pathway in which phosphatidylinositol-4-phosphate (PI-4-P) is the intermediate. Here we examined the effect of activation of the PI-5-P pathway on phosphoinositide 3-kinase (PI3K) signaling by expressing PIP4K II beta in cells that lack this enzyme. Although PIP4K II generates PI-4,5-P2, a substrate for PI3K, expression of this enzyme reduced rather than increased phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) levels in cells stimulated with insulin or cells expressing activated PI3K. This reduction in PI-3,4,5-P3 levels resulted in decreased activation of the downstream protein kinase, Akt/PKB. Consistent with these results, expression of IpgD, a bacterial phosphatase that converts PI-4,5-P2 to PI-5-P, resulted in Akt activation, and this effect was partially reversed by PIP4K II beta. PIP4K II beta expression did not impair insulin-dependent association of PI3K with insulin receptor substrate 1 (IRS1) but abbreviated Akt activation, indicating that PIP4K II regulates PI-3,4,5-P3 degradation rather than synthesis. These data support a model in which the PI-5-P pathway controls insulin signaling that leads to Akt activation by regulating a PI-3,4,5-P3 phosphatase.     

Myocardial alpha 1-adrenoceptors mediate positive inotropic effect and changes in phosphatidylinositol metabolism. Species differences in receptor distribution and the intracellular coupling process in mammalian ventricular myocardium.

Species-dependent variations of myocardial alpha 1-adrenoceptor-mediated positive inotropic effects of epinephrine were assessed in relation to characteristics of alpha 1-receptor bindings and acceleration of phosphatidylinositol metabolism in the isolated rat, rabbit, and dog ventricular myocardium. Epinephrine in the presence of the beta-adrenoceptor antagonist bupranolol (10(-6) M) elicited a positive inotropic effect through activation of alpha 1-adrenoceptors in rat and rabbit, whereas in dog ventricular myocardium, bupranolol abolished the positive inotropic effect of epinephrine. [3H]Prazosin bound to membrane fractions derived from rat, rabbit, and dog ventricular muscle with high affinities in a saturable and reversible manner. In dog, Bmax and Kd values of alpha 1-adrenoceptor binding sites were identical to those in rabbit ventricular muscle. The Bmax value of alpha 1-adrenoceptors in rat ventricle was the highest, amounting to two to four times those in rabbit and dog. Epinephrine displacement curves for the specific binding of [3H]prazosin in the membrane fraction of these species showed high and low affinity sites with slope factors significantly less than unity, which were shifted to single low affinity sites with slope factors close to unity by addition of 5'-guanylylimidodiphosphate. Accumulation of [3H]inositol 1-phosphate [( 3H]IP1) in ventricular slices prelabeled with [3H]myo-inositol was increased by epinephrine in a time- and concentration-dependent manner in rat ventricular slices. [3H]IP1 accumulation likewise was facilitated by alpha 1-adrenoceptor stimulation in rabbit ventricular slices, whereas the extent of [3H]IP1 accumulation was much less than that in rat. In dog ventricular slices, [3H]IP1 was not accumulated by epinephrine. In rabbit papillary muscle, the time course of increase in contractile force induced by alpha-adrenoceptors coincided with the prolongation of the action potential duration with a similar time course, which is in strong contrast to previous findings in rat that the contractile response was dissociated from the electrophysiological response to alpha-adrenoceptor stimulation. The present results indicate that a wide range of variation of alpha 1-adrenoceptor-mediated regulation of myocardial contractility may be ascribed to different contributions of facilitatory as well as inhibitory regulatory processes that lead to intracellular Ca2+ mobilization subsequent to myocardial alpha 1-adrenoceptor activation among mammalian species.     

Interaction of alpha1-adrenoceptor subtypes with different G proteins induces opposite effects on cardiac L-type Ca2+ channel

We examined the effect of alpha(1)-adrenoceptor subtype-specific stimulation on L-type Ca2+ current (I(Ca)) and elucidated the subtype-specific intracellular mechanisms for the regulation of L-type Ca2+ channels in isolated rat ventricular myocytes. We confirmed the protein expression of alpha(1A)- and alpha(1B)-adrenoceptor subtypes at the transverse tubules (T-tubules) and found that simultaneous stimulation of these 2 receptor subtypes by nonsubtype selective agonist, phenylephrine, showed 2 opposite effects on I(Ca) (transient decrease followed by sustained increase). However, selective alpha(1A)-adrenoceptor stimulation (> or =0.1 micromol/L A61603) only potentiated I(Ca), and selective alpha(1B)-adrenoceptor stimulation (10 mumol/L phenylephrine with 2 micromol/L WB4101) only decreased I(Ca). The positive effect by alpha(1A)-adrenoceptor stimulation was blocked by the inhibition of phospholipase C (PLC), protein kinase C (PKC), or Ca2+/calmodulin-dependent protein kinase II (CaMKII). The negative effect by alpha(1B)-adrenoceptor stimulation disappeared after the treatment of pertussis toxin or by the prepulse depolarization, but was not attributable to the inhibition of cAMP-dependent pathway. The translocation of PKCdelta and epsilon to the T-tubules was observed only after alpha(1A)-adrenoceptor stimulation, but not after alpha(1B)-adrenoceptor stimulation. Immunoprecipitation analysis revealed that alpha(1A)-adrenoceptor was associated with G(q/11), but alpha(1B)-adrenoceptor interacted with one of the pertussis toxin-sensitive G proteins, G(o). These findings demonstrated that the interactions of alpha(1)-adrenoceptor subtypes with different G proteins elicit the formation of separate signaling cascades, which produce the opposite effects on I(Ca). The coupling of alpha(1A)-adrenoceptor with G(q/11)-PLC-PKC-CaMKII pathway potentiates I(Ca). In contrast, alpha(1B)-adrenoceptor interacts with G(o), of which the betagamma-complex might directly inhibit the channel activity at T-tubules.     

Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes.

alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.     

Alpha(1)-adrenoceptor subtypes mediate negative inotropy in myocardium from alpha(1A/C)-knockout and wild type mice

Cardiac alpha(1)-adrenoceptors (AR) have two predominant subtypes (alpha(1A)-AR and alpha(1B)-AR) however, their roles in regulating contraction are unclear. We determined the effects of stimulating alpha(1A)-AR (using the subtype-selective agonist A61603) and alpha(1B)-AR (using a gene knockout mouse lacking alpha(1A)-AR) separately, and together (using phenylephrine) on Ca(2+) transients, intracellular pH, and contraction of mouse cardiac trabeculae. Stimulation of alpha(1)-AR subtypes separately or together caused a triphasic contractile response. After a transient ( approximately 3%) force rise (phase 1), force declined markedly (phase 2), then partially recovered (phase 3). In phase 2, the force decline (% of initial) with combined alpha(1A)-AR plus alpha(1B)-AR stimulation (50+/-3%) was more than with separate subtype stimulation (P<0.01), suggesting alpha(1A)-AR and alpha(1B)-AR mediate additive effects during phase 2. Force decline in phase 2 paralleled decreases of Ca(2+) transients that were reduced more with combined vs. separate subtype stimulation. During phase 3 the final force reduction was similar with stimulation of alpha(1A)-AR (20+/-5%), or alpha(1B)-AR (20+/-3%), or both (26+/-4%) suggesting alpha(1A)-AR and alpha(1B)-AR mediate non-additive effects during phase 3. In contrast, Ca(2+) transients recovered fully in phase 3 suggesting reduced force in phase 3 involved decreased myofilament Ca(2+)-sensitivity. Decreased Ca(2+)-sensitivity was not mediated by changes of intracellular pH since this was not affected by alpha(1)-AR stimulation. In contrast to mouse trabeculae, rat trabeculae demonstrated a positive inotropic response to alpha(1)-AR stimulation. In conclusion, for mouse myocardium in vitro both alpha(1)-adrenoceptor subtypes mediate negative inotropy involving decreased Ca(2+) transients and a decreased Ca(2+) sensitivity that does not involve altered intracellular pH.     

Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis

To produce blood platelets, megakaryocytes elaborate proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. The invaginated demarcation membrane system (DMS), a hallmark of mature cells, has been proposed as the source of proplatelet membranes. By direct visualization of labeled DMS, we demonstrate that this is indeed the case. Late in megakaryocyte ontogeny, the DMS gets loaded with PI-4,5-P(2), a phospholipid that is confined to plasma membranes in other cells. Appearance of PI-4,5-P(2) in the DMS occurs in proximity to PI-5-P-4-kinase alpha (PIP4Kalpha), and short hairpin (sh) RNA-mediated loss of PIP4Kalpha impairs both DMS development and expansion of megakaryocyte size. Thus, PI-4,5-P(2) is a marker and possibly essential component of internal membranes. PI-4,5-P(2) is known to promote actin polymerization by activating Rho-like GTPases and Wiskott-Aldrich syndrome (WASp) family proteins. Indeed, PI-4,5-P(2) in the megakaryocyte DMS associates with filamentous actin. Expression of a dominant-negative N-WASp fragment or pharmacologic inhibition of actin polymerization causes similar arrests in proplatelet formation, acting at a step beyond expansion of the DMS and cell mass. These observations collectively suggest a signaling pathway wherein PI-4,5-P(2) might facilitate DMS development and local assembly of actin fibers in preparation for platelet biogenesis.     

衰老大鼠心脏血管紧张素Ⅱ受体对α_1肾上腺素受体正性变力效应的影响

目的研究激动血管紧张素Ⅱ(AngⅡ)1型受体(AT1R)和2型受体(AT2R)对α1肾上腺素受体(α1-AR)介导的心肌正性变力效应的影响,以及这种影响在大鼠心脏衰老过程中的变化。方法对于3.5、12、18、24月龄的Wistar大鼠采用体外左心房收缩功能实验,观察AngⅡ分别激动AT1R和AT2R对苯肾上腺素激动α1-AR介导正性变力效应的影响。结果在AT2R阻滞剂PD123319存在时,AngⅡ激动AT1R,与AngⅡ共同激动AT1R和AT2R时比较,3.5、12月龄大鼠α1-AR介导的正性变力效应的最大收缩效应(Rmax)及Rmax50%时的药物浓度值(以其负对数pD2值表示)差异均无显著性意义;18、24月龄大鼠Rmax及pD2值均增大。在AT1R阻滞剂氯沙坦存在时,AngⅡ激动AT2R,与AngⅡ共同激动AT1R和AT2R时比较,各月龄大鼠α1-AR介导的正性变力效应Rmax及pD2值差异均无显著性意义。结论随着鼠龄的增长,激动AT1R可增强大鼠心肌α1-AR介导的正性变力效应,而激动AT2R对α1-AR介导的心肌正性变力效应没有影响。     

Effects of alpha1-adrenergic stimulation on normal and hypertrophied mouse hearts. Relation to caveolin-3 expression

BACKGROUND: Modulation of the transduction efficiency through G-protein coupled receptors, caused by external stimulation, is essential in designing antihypertrophic treatment strategies in the dysfunctional heart. We compared protein-kinase C (PKC)-dependent regulation of positive inotropic effect via alpha1-adrenoreceptor (ADR)/Gq protein in hyperdynamic versus hypertrophied myocardium. METHODS: Inotropic (work performing isolated heart) and cellular effects of alpha1-adrenoreceptor stimulation were studied in nontransgenic (Ntg) and transgenic (Tg) mice with cardiac specific overexpression of L-type voltage-dependent calcium channels (L-type VDCC). RESULTS: Transgenic hyperdynamic and hypertrophic myocardium (due to overexpression of the L-type VDCC alpha1 subunit) were characterized by a lack of positive inotropic effect (PIE) to alpha1-ADR stimulation with phenylephrine (PE), as compared to a positive response in Ntg hearts. This was partially restored by PKC inhibition with chelerythrine and staurosporine only at the hyperdynamic stage. The inability of PKC inhibition to increase positive inotropy was associated with markedly decreased cardiac-specific caveolin-3 expression, and no changes in Galphaq, PLC-beta1, caveolin-1 and alpha1-adrenoreceptor expression. CONCLUSION: In the hyperdynamic myocardium, PKC activation may be one of the switches responsible for an impaired alpha1-adrenergic positive inotropic response. In the hypertrophied myocardium, the interruption of the transduction from Galphaq-protein coupled receptors to downstream effectors may be due to the down-regulation of caveolin-3 expression.     

Influence of calcium-entry blockade on vasoconstrictor responses in feline mesenteric vascular bed

The subtypes of postjunctional alpha-adrenoceptors activated by neuronally released and exogenous norepinephrine and the source of calcium used for vasoconstrictor responses were investigated in the feline mesenteric vascular bed. Under constant flow conditions, intra-arterial injections of phenylephrine and UK14304, alpha 1- and alpha 2-adrenoceptor agonists, increased mesenteric arterial perfusion pressure in a dose-related manner. Prazosin, an alpha 1-antagonist, reduced vasoconstrictor responses to phenylephrine without altering responses to UK14304. Yohimbine, an alpha 2-antagonist, reduced responses to UK14304 without altering responses to phenylephrine. The same pattern of blockade was observed in animals pretreated with 6-hydroxydopamine to destroy the integrity of adrenergic terminals. Responses to phenylephrine and UK14304 were reduced by nitrendipine, a calcium-entry blocking agent, and this agent decreased vasoconstrictor responses to sympathetic nerve stimulation, tyramine, and norepinephrine. Responses to sympathetic nerve stimulation were selectively blocked by prazosin, but responses to norepinephrine were selectively blocked by yohimbine. Vasoconstrictor responses to tyramine were reduced by both prazosin and yohimbine. Nitrendipine also reduced responses to angiotensin II, U46619, a prostaglandin endoperoxide analogue, Bay K 8644, and potassium chloride. These data suggest the presence of alpha 1- and postjunctional alpha 2-adrenoceptors and support the hypothesis that norepinephrine released by nerve excitation acts mainly on alpha 1-receptors but that exogenous norepinephrine acts primarily on alpha 2-receptors. However, norepinephrine released by tyramine acts on both receptor subtypes. Nitrendipine inhibited responses to the alpha 1- and alpha 2-adrenoceptor agonists as well as those to nerve released and exogenous norepinephrine, the calcium agonist, Bay K 8644, and to other vasoconstrictor agents. These data suggest that in the feline mesenteric vascular bed, an extracellular source of calcium ions is required for vasoconstriction induced by a variety of mechanisms including activation of alpha 1- and postjunctional alpha 2-adrenoceptors.     

Effects of alpha 1- and alpha 2-adrenoceptor stimulation and blockade on plasma insulin levels in the mouse

Stimulation of alpha-adrenoceptors is known to inhibit insulin secretion under a variety of conditions. In this study, the question of whether these alpha-adrenoceptors are of the alpha 1- or the alpha 2-subtype was investigated in the mouse. The selective alpha 2-adrenoceptor agonist clonidine (0.05-50 nmol/kg) was found to markedly inhibit the insulin secretory response to both glucose and the cholinergic agonist carbachol. This inhibition of insulin secretion was counteracted by the alpha 2-adrenoceptor antagonist yohimbine (2.6 mumol/kg), but not by the alpha 1-adrenoceptor antagonist prazosin (2.6 mumol/kg). In contrast, the alpha 1-adrenoceptor agonist phenylephrine (0.05-50 nmol/kg) did not affect the insulin secretory response to either glucose or carbachol. Moreover, both yohimbine and prazosin increased basal plasma insulin levels. It is concluded that alpha 1- and alpha 2-adrenoceptor blockade is followed by enhancement of basal plasma insulin levels in the mouse, whereas alpha 2-adrenoceptor stimulation but not alpha 1-adrenoceptor stimulation impairs the insulin secretory response to glucose and carbachol.     

Decrease in alpha 1-adrenoceptor reserve in mesenteric arteries isolated from spontaneously hypertensive rats

The characterization of alpha-adrenoceptor-mediated contractile responses and the effects of the calcium channel blocker nifedipine on these responses were investigated in mesenteric arterial strips from 13-week-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Contractile responses to the alpha-adrenoceptor agonists phenylephrine and clonidine were mediated through the activation of alpha 1-adrenoceptors. The dose producing a half-maximum response (ED50) for the agonists was higher in SHR than in WKY. Affinities of alpha 1-adrenoceptors were similar between the two strains. When arterial strips from both strains were treated with the same concentration of phenoxybenzamine, the maximum response to each agonist was weaker in SHR. The alpha 1-adrenoceptor occupancy-response relationship for phenylephrine was hyperbolic and less steep in SHR, while the relationship for clonidine was linear in SHR but not in WKY. Alpha 1-adrenoceptor occupancy at a half-maximum response to each agonist was greater in SHR. Nifedipine inhibited the maximum responses to the agonists more profoundly in SHR than in WKY. This inhibition was greater in the response to clonidine than in the response to phenylephrine in both strains. When the maximum response to phenylephrine was reduced to the same extent in both strains by treatment with different concentrations of phenoxybenzamine, the responses to phenylephrine were more susceptible to inhibition by nifedipine. Under these conditions, the effects of nifedipine were similar between SHR and WKY. These results suggest that alpha 1-adrenoceptor reserve is reduced in SHR mesenteric artery compared with WKY, which may be responsible for the greater inhibition by nifedipine of the alpha 1-adrenoceptor-mediated contractions in SHR.     

Peripubertal changes in the nature of the GnRH response to alpha-adrenoceptor stimulation in vitro and their modulation by testosterone

Adrenergic mechanisms have been widely implicated in the regulation of GnRH secretion in adult rats but their role in young animals, in which the activity of the GnRH neurones is minimal, is unclear. These experiments were done to examine the effects of alpha-adrenoceptor stimulation on the secretion in vitro of GnRH by hypothalami from immature and adult male rats. The alpha 1-adrenoceptor agonist, phenylephrine (10(-9) - 10(-7) M), stimulated release of GnRH from hypothalami from adult (200 g) and peripubertal (150 g) rats but inhibited markedly the secretion of the releasing factor from the limited stores available in hypothalami from immature (50 or 100 g) rats. The stimulatory and inhibitory responses to phenylephrine, evident in adult and younger rats respectively, were concentration-dependent and antagonized readily by the selective alpha 1-adrenoceptor antagonist, alfuzosin (10(-6) M), but not by the beta-adrenoceptor antagonist, propranolol (10(-6) M). Hypothalami from 14-day castrated adult rats, in which the serum LH was elevated and hypothalamic GnRH content reduced, responded to alpha 1-adrenoceptor stimulation in vitro, like those from immature rats, with a marked reduction in GnRH release. In contrast, hypothalami from corresponding castrates bearing testosterone implants, which maintained the hypothalamic GnRH content and serum LH and testosterone concentrations at levels similar to those of intact controls, exhibited the normal 'adult' response to phenylephrine. Studies utilizing 3H-prazosin indicated that the number (Bmax) of hypothalamic alpha 1-adrenoceptor binding sites increases at puberty but that receptor affinity (KD) is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C delta and epsilon mediate positive inotropy in adult ventricular myocytes

To examine cardiac contractile regulation and protein kinase C (PKC) translocation in parallel, the delta and epsilon isoforms of PKC were fused to green fluorescent protein (GFP) and expressed in adult rat ventricular myocytes maintained in short term culture. PKC-delta-GFP and PKC-epsilon-GFP were predominantly cytosolic until phorbol dibutyrate (PDBu) was introduced. PKC-delta-GFP redistributed preferentially to perinuclear structures that co-localized with a Golgi marker, whereas PKC-epsilon-GFP redistributed preferentially to the surface sarcolemma. Myocyte contractile function was assessed by monitoring twitch shortening with field stimulation at 0.5 Hz, 22 degrees C. In myocytes expressing PKC-delta-GFP, PDBu caused a transient negative inotropic response followed by a robust and sustained positive inotropic response that paralleled perinuclear PKC-delta accumulation. In PKC-epsilon-GFP myocytes, PDBu caused a sustained negative inotropic response that paralleled accumulation at the surface sarcolemma, but this response did not differ from myocytes expressing GFP alone. At higher expression levels, PKC-epsilon-GFP myocytes responded more like PKC-delta-GFP myocytes including perinuclear accumulation and a sustained positive inotropic response. Positive inotropic responses were markedly attenuated if PKC translocation was biased toward the surface sarcolemma by use of a more hydrophobic PKC activator, and were completely and selectively blocked by the PKC antagonist bis-indoylmaleimide. In contrast, transient and sustained negative inotropic responses were selectively blocked by the Ca(2+)-dependent PKC isoform antagonist Go6976. The data indicate that the novel PKC isoforms delta and epsilon have little effect on contractility when accumulating at the cell surface, but produce a strong positive inotropic response upon accumulation at the Golgi or other intracellular sites.     

Alpha 1-adrenoceptor stimulation enhances the delayed rectifier K+ current of guinea pig ventricular cells through the activation of protein kinase C.

The effect of alpha 1-adrenoceptor stimulation on the delayed rectifier K+ current (IK) was examined in isolated guinea pig ventricular cells by use of the patch-clamp method. IK was evoked by a 3-second depolarizing pulse from a holding potential of -30 mV in a Na(+)- and K(+)-free solution containing 3 microM nifedipine. Phenylephrine (30 microM) in the presence of propranolol (1 microM) produced an increase in IK. In five cells, phenylephrine increased the tail current of IK by 23 +/- 5%. This effect of phenylephrine was blocked by prazosin (0.3 microM), a selective alpha 1-blocker. Phenylephrine produced only a small effect on the voltage and time dependence of IK. Pretreatment with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 10 microM) abolished the phenylephrine-induced increase in IK. In addition, pretreatment with a maximally effective concentration of 12-O-tetradecanoylphorbol 13-acetate (100 nM) abolished the phenylephrine-induced increase in IK. In conclusion, alpha 1-adrenoceptor stimulation increases IK in guinea pig cardiomyocytes. This alpha 1-adrenoceptor-mediated response may be related to an activation of protein kinase C. The increase in IK may explain a shortening of action potential duration observed after alpha 1-adrenoceptor stimulation in guinea pig cells.     

Angiotensin-stimulated production of inositol trisphosphate isomers and rapid metabolism through inositol 4-monophosphate in adrenal glomerulosa cells.

The production and metabolism of inositol phosphates in rat adrenal glomerulosa cells prelabeled with [3H]inositol and stimulated with angiotensin II were analyzed by high-performance anion-exchange chromatography. Exposure to angiotensin II was accompanied by a rapid and substantial decrease in the phospholipid precursor, phosphatidylinositol (PtdIns) 4,5-bisphosphate with only a slight and transient increase in the level of the biologically active product, inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), to a peak at about 5 sec. Inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), the putative metabolite of Ins-1,4,5-P3, was also formed rapidly and maintained an elevated steady-state level during stimulation by angiotensin II. Inositol 1,4-bisphosphate (Ins-1,4-P2) exhibited a simultaneous and prominent increase that could not be accounted for solely by direct breakdown of PtdIns 4-phosphate, indicating that large amounts of Ins-1,4,5-P3 must also have been produced and metabolized. The rapid formation of a substantial amount of inositol 4-monophosphate (Ins-4-P), with no significant change in the level of inositol 1-monophosphate (Ins-1-P) during the first minute of stimulation, was a notable feature of the glomerulosa cell response to angiotensin II. These observations indicate that PtdIns-4,5-P2 catabolism in the angiotensin-stimulated glomerulosa cell initially proceeds via Ins-1,4,5-P3 through Ins-1,3,4-P3 and Ins-1,4-P2 to form Ins-4-P rather than Ins-1-P and that direct hydrolysis of PtdIns by phospholipase C does not occur during the initial phase of angiotensin action. In glomerulosa cells stimulated by angiotensin II in the presence of Li+, the progressive accumulation of both Ins-4-P, and after a short lag period, Ins-1-P indicated that dephosphorylation of both isomers was inhibited by Li+. The increase of Ins-P isomers in the presence of Li+ was associated with increased and progressive accumulation of Ins-1,4-P2 and Ins-1,3,4-P3 but not of Ins-1,4,5-P3. These data demonstrate that sustained and massive breakdown of PtdIns phosphates begins within seconds during cell activation by angiotensin II. The Ca2+-mobilizing metabolite, Ins-1,4,5-P3, is rapidly converted to Ins-1,3,4-P3 and degraded through Ins-1,4-P2 and Ins-4-P, in contrast to the previous view that conversion to Ins-1-P is the major route of PtdIns 4,5-bisphosphate metabolism.     

Effects of alpha-adrenoceptor agonists and antagonists on thyroid hormone secretion

The effects of various alpha-adrenoceptor agonists and antagonists on blood radioiodine levels were studied in mice pre-treated with 125I and thyroxine. The non-selective alpha-adrenoceptor agonist noradrenaline and the selective alpha 1-adrenoceptor agonist phenylephrine both enhanced blood radioiodine levels. Noradrenaline was more potent than phenylephrine. Contrary, the selective alpha 2-adrenoceptor agonist clonidine depressed basal levels of blood radioiodine. The non-selective alpha-adrenoceptor antagonist phentolamine and the selective alpha 1-adrenoceptor antagonist prazosin both inhibited the noradrenaline-induced elevation of radioiodine levels, whereas the alpha 2-adrenoceptor antagonist yohimbine had no such effect, except at a high dose level. All three alpha-adrenoceptor agonists, noradrenaline, phenylephrine and clonidine, inhibited the radioiodine response to TSH. In addition, TSH-induced increase in radioiodine levels was inhibited by prazosin, whereas yohimbine had no effect. Phentolamine inhibited the radioiodine response to TSH when given 2 h prior to TSH, whereas when given 15 min prior to TSH the response to TSH was potentiated by phentolamine. It is concluded, that under in vivo conditions in the mouse, alpha 1-adrenoceptor activation stimulates basal thyroid hormone secretion and inhibits TSH-induced thyroid hormone secretion. Further, alpha 2-adrenoceptor activation inhibits basal thyroid hormone secretion. In addition, TSH-induced thyroid hormone secretion is inhibited by alpha 1-adrenoceptor antagonism. Thus, alpha-adrenoceptors induce both stimulatory and inhibitory effects of thyroid function.     

Role of the inositol phosphate cycle in the cardioprotective effect of adaptation to intermittent hypoxia]

The impact of adaptation to intermittent hypoxia of different duration (for 20 and 40 days) on the inositol triphosphate-diacylglycerol (ITP-DAG) regulatory contour in the heart was investigated. For this, isolated heart alpha 1-adrenoreactivity to phenylephrine and phospholipase (PhL-C) activities were studied in cardiac plasma membranes. On day 20 of adaptation, the heart inotropic response to phenylephrine stimulation was somewhat reduced and the activity of Ca(2+)-dependent PhL-C did not differ from that in the controls within the range of Ca2+ physiological concentrations. Forty days after adaptation, both positive inotropic responses of the heart to phenylephrine and activity of Ca(2+)-dependent PhL-C were enhanced, i.e. activation of the ITP-DAG regulatory cascade occurred. Early studies have shown that a phenomenon of adaptive stabilization develops at this stage of adaptation. The phenomenon appears as a significant increase in heart resistance to thermal injury, toxic catecholamine levels, Ca2+ paradox and high Ca2+ concentrations. Thus, the revealed activation of the ITP-DAG regulatory contour was accompanied by the development of a phenomenon of adaptive structure stabilization (PhASS) in the hearts of adapted animals. The findings suggest that the ITP-DAG regulatory cascade plays an important role in the cardioprotective effect of adaptation to intermittent hypobaric hypoxia and in PhASS formation.     

The ?3-adrenoceptor and its regulation in cardiac tissue

In addition to beta1 and beta2-adrenoceptors, the recently cloned beta3-adrenoceptor is also expressed at the surface of myocardial and vascular cells, albeit with considerable variability among species. In human ventricular muscle, stimulation of this receptor produces a negative inotropic effect that involves, at least in part, activation of the endothelial nitric oxide synthase (eNOS) through G-alpha-i proteins, and intracellular increases in cyclic GMP. In the non-failing heart, this beta3-adrenoceptor pathway may protect the myocardium against the toxic effects of excessive stimulation by catecholamines. In biopsy samples from human failing ventricular myocardium (from ischemic, dilated or septic cardiomyopathies), the abundnace of beta3-adrenoceptor proteins is increased, as that of the coupling G-alpha-i proteins. In the setting of high orthosympathetic input to the heart, catecholamine stimulation of the poorly desentitizable beta3-adrenoceptor, combined with desensitized/downregulated beta1 and beta2-adrenaceptors, may favor a sustained and prevailing beta3-adrenergic negative inotropic effect. The pathophysiological importance of this pathway in the clinical syndrome of heart failure will await the result of trials with antagonists specific for the human cardiac beta3-adrenoceptor.