磷酸二酯酶IV在心血管系统中的表达、活性及作用

磷酸二酯酶(PDEs)催化水解细胞内第二信使cAMP及cGMP,使细胞内cAMP和cGMP呈浓度梯度及区室化分布,在信号传导和调控生理学反应中起中心作用,包括心血管系统在内的所有类型细胞的新陈代谢、收缩性、运动性和转录等。PDE4是cAMP特异性的水解酶,其水解活性通常占细胞内水解cAMP活性的绝大部分。近来的研究表明,PDE4在心血管系统的各种细胞类型上都有表达,在调控心血管系统的运动中有重要的作用,其表达和活性的改变与心衰和心律失常的发展有关。这些研究为开发治疗心血管疾病的药物提供了新的靶点。     

Inhibition of cardiovascular low-Km cAMP phosphodiesterase activity by medorinone

Medorinone (5-methyl-1, 6-naphthyridin-2(1H)-one) is a structurally novel cardiotonic/vasodilator agent. The purpose of the present study was to evaluate the effects of medorinone on subcellular systems involved in the contractile-relaxation processes of cardiac and vascular smooth muscle (VSM); cyclic nucleotide phosphodiesterase (PDE), Ca²⁺-regulated contractile proteins, and sarcolemmal Na⁺-K⁺ ATPase. Medorinone produces concentration-related inhibition (IC₅₀ values between 0.38 and 1.3 μ) of the cGMP-inhibitable, low-K_m cAMP PDE (peak III PDE from a DEAE-cellulose column) present in guinea pig and canine cardiac and vascular smooth muscle. The potency of medorinone is similar to that of milrinone and papaverine, less than CI-930, and greater than imazodan, piroximone, and amrinone. Medorinone is approximately 300–400 × less potent an inhibitor of the low-K_m cAMP PDE. Medorinone appears to be a non-linear mixed inhibitor of the low-K_m cAMP PDE having a K_i value of approximately 0.3 μM. Medorinone is also an equipotent inhibitor (IC₅₀ values between 0.23 and 0.35 μM) of the low-_m cAMP PDE present in soluble and particulate fractions obtained from normal and myopathic human hearts, with no differences in potency observed between normal and myopathics. Medorinone (10 and 100 μM) does not affect VSM myosin light chain phosphorylation or VSM actomyosin superprecipitation. Moreover, medorinone does not affect cardiac myofibrillar ATPase activity or cardiac sarcolemmal Na⁺-K⁺ ATPase activity. In summary, these data are consistent with the hypothesis that inhibition of the low-K_m cAMP PDE isozyme by medorinone in cardiac and vascular smooth muscle is a mechanism by which this agent produce both positive cardiac inotropy and VSM relaxation.     

Selective inhibition of cAMP phosphodiesterase III activity by the cardiotonic agent saterinone in guinea pig myocardium

The effects of saterinone [+/-)-1,2-dihydro-5-[4-[2-hydroxy-3-[4-(2-methoxyphenyl)-1-piperazinyl] propoxy] phenyl]-6-methyl-2-oxo-3-pyridine-carbonitrile, BDF 8634) on force of contraction, beating frequency, and on phosphodiesterase (PDE) activity were investigated in isolated preparations from guinea pig hearts. The effects of 3-isobutyl-1-methylxanthine (IBMX) and milrinone were studied for comparison. Saterinone exerted a concentration-dependent (1-30 mumol/l) positive inotropic effect (EC50 = 9.1 mumol/l) in guinea pig papillary muscles. The drug was more potent in increasing force of contraction than was milrinone, but its efficacy was only half as great as that of milrinone. Also IBMX revealed an about 4-fold greater positive inotropic efficacy than saterinone. This study provides functional evidence that the positive inotropic effect of saterinone is at least partially due to a cyclic adenosine monophosphate (cAMP)-dependent mechanism because carbachol antagonized the increase in force of contraction completely. In order to elucidate a possible mechanism of action of saterinone its effects on cardiac cAMP PDE activity were investigated. Saterinone selectively and potently inhibited the isoenzyme III of PDE (IC50 = 0.06 mumol/l). The mean IC50-values for the inhibition of PDE I and II were 323-fold greater. Thus, saterinone proved to be a selective inhibitor of "low-Km, cAMP specific" PDE III. Saterinone revealed only a moderate positive chronotropic effect. The beating frequency of guinea pig spontaneously beating right auricles was augmented by 26.4% at most, that is half as much as the effect of isoprenaline at a similarly effective positive inotropic concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The differential effect of lithium on noradrenaline- and dopamine-sensitive accumulation of cyclic AMP in guinea pig brain

Lithium (Li) in its narrow therapeutic concentration range was found to inhibit only the noradrenaline- and not the dopamine-sensitive accumulation of cyclic AMP in guinea pig brain. The results suggest a pharmacological distinction between the antischizophrenic drugs that inhibit dopamine-sensitive cyclic AMP accumulation and Li, an antimanic agent that inhibits specifically only the noradrenalinesensitive cyclic AMP accumulation.     

Synaptosomal (Ca2+-Mg2+)-ATPASE activity modulation by cyclic AMP

Dibutyryl cyclic AMP, in a concentration-dependent manner, increased synaptosomal (Ca2+-Mg2+)-ATPase activity, but in synaptic plasma membranes lacked any effect. The maximal enzyme activity in synaptosomes was increased by 38%, leaving unaltered the extrasynaptosomal Ca2+ concentration necessary to reach it. In the presence of 5 microM cyclic AMP, cyclic AMP-dependent protein kinase increased (30%) maximal (Ca2+-Mg2+)-ATPase activity in synaptic plasma membranes, but the apparent affinity for Ca2+ was not modified. This effect was partially inhibited (60%) by a cyclic AMP-dependent protein kinase inhibitor. The data suggest that synaptosomal (Ca2+-Mg2+)-ATPase activity is modulated by a cyclic AMP-dependent phosphorylation reaction.     

Ca^2＋ participates in α1B-adrenoceptor-mediated cAMP response in HEK293 cells

AIM: To investigate the alpha1B-adrenoceptor (alpha1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells. METHODS: Full-length cDNA encoding alpha1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and alpha1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively. RESULTS: Under agonist stimulation, alpha1B-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin (PTX) had little effect on basal cAMP accumulation as well as norepinephrine (NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin (CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdCl2 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59%+/-0.47% to 1.00%+/-0.31%, 0.78%+/-0.23%, and 0.90%+/-0.40%, respectively. CONCLUSION: By coupling with a PTX-insensitive G protein, alpha1BAR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC), thereby increasing the cAMP production in HEK293 cell lines.     

异丙肾上腺素及氨茶碱通过cAMP诱导的高电导Ca^2＋激活钾通道活化舒张支气管平滑肌

目的：探讨异丙肾上腺素(Iso)和氨茶碱(Ami)是否通过蛋白激酶A(PKA)通道激活高电导Ca^2 活化钾通道(BKCa)来舒张支气管平滑肌．方法：运用等长张力记录和穿孔膜片箝技术,观察Iso和Ami对大鼠离体支气管平滑肌细胞BKCa的作用以及PKA抑制剂Rp—cAMP对该效应的影响．结果：(1)Iso和Ami均可诱发甲酰胆碱预收缩的离体支气管产生浓度依赖性舒张反应,BKCa阻断剂四乙胺(TEA)5mmol/L使两者的量效曲线向右显著移位;(2)Iso 1 μmol/L显著增加方波刺激时(从-60mV到 50mV)平滑肌细胞BKCa电流,该效应可被Rp—cAMP l00μmol/L显著抑制,Iso使BKCa电流电压关系曲线向上移位．在斜坡刺激时(从-100mV到 100mV)亦获得类似结果;(3)Ami 1 mmol/L显著增加支气管平滑肌细胞在方波刺激时的BKCa电流,该效应被Rp—cAMP l00μmol/L显著抑制,Ami使BKCa电流电压关系曲线向上移位．在斜坡刺激时亦获得类似结果．结论：cAMP依赖性的气道舒张剂Iso和Ami对大鼠气道的舒张效应至少部分通过PKA的活化激活BKCa通道而实现的．     

Selective inhibitory effect of new phosphodiesterase inhibitors on PDE isozymes in guinea pig cardiac muscle

Selective inhibition of seven new PDE inhibitors on cyclic nucleotide PDE isozymes was investigated. Three PDE isozymes (PDE I, II and III) of guinea pig left ventricular muscles were used. All tested agents inhibited cyclic AMP hydrolysis by PDE III in a concentration-dependent manner. Some agents represented more potent and selective inhibitory effect on PDE III than that of imazodan.     

The effects of amrinone on contractility, Ca2+ uptake and cAMP in smooth muscle

Amrinone, a known positive inotropic agent in the heart, was found to cause a dose-dependent (10--100 micrograms/ml) inhibition of norepinephrine (NE) or high-K+-induced contractions of rabbit aorta. Amrinone also inhibited carbachol or high-K+-induced contractions of guinea-pig taenia coli. Neither total tissue 45Ca uptake nor the rate of 45Ca uptake induced by 80 mM K+ in rabbit aorta was altered by pretreatment with amrinone. On the other hand, a similar pretreatment with amrinone inhibited NE (10(-6) or 10(-5) M) induced tissue 45Ca uptake. Amrinone (100 micrograms/ml) caused about a 70% increase in cAMP concentration over resting levels. It is concluded that amrinone causes a nonspecific inhibition of smooth muscle contractility by acting probably at multiple sites to decrease the availability of Ca2+ required for activation. One or more of these mechanisms may involve cAMP.     

Characterization of phosphodiesterase 4 in guinea-pig macrophages: multiple activities,association states and sensitivity to selective inhibitors

1. The cyclic AMP phosphodiesterases (PDE) in guinea-pig peritoneal macrophages were isolated, partially characterized and their role in regulating the cyclic AMP content in intact cells evaluated.
2. Differential centrifugation of macrophage lysates revealed that ∼90% of the PDE activity was membrane-bound and exclusively hydrolyzed cyclic AMP. This activity was not removed by KCl (200 mM) but was readily solubilized by the non-ionic detergent, Triton X-100 (1% v/v). Greater than 80% of the hydrolytic activity was suppressed by the PDE4 inhibitors, R-rolipram and nitraquazone with IC₅₀s of 240 and 540 nM, respectively.
3. Anion-exchange chromatography of the total protein extracted from macrophages resolved two major peaks of cyclic AMP PDE activity that were insensitive to cyclic GMP (10 μM), calmodulin (50 units plus 2 mM CaCl2) and a PDE3 inhibitor, SK&F 95654 (10 μM), but were markedly suppressed by RS-rolipram (10 μM). The two peaks of PDE activity were arbitrarily designated CPPDE4α and CPPDE4β with respect to the order from which they were eluted from the column where the prefix, CP, refers to the species, Cavia porcellus.
4. The hydrolysis of cyclic AMP catalyzed by CPPDE4α and CPPDE4β conformed to Michaelis-Menten kinetic behaviour with similar K_ms (13.4 and 6.4 μM, respectively).
5. Thermal denaturation of membrane-bound PDE4 at 50°C followed bi-exponential kinetics with t_1/2 values of 1.5 and 54.7 min for the first and second components, respectively. In contrast, CPPDE4α and CPPDE4β each decayed mono-exponentially with significantly different thermostabilities (t_1/2=2.77 and 1.15 min, respectively).
6. Gel filtration of CPPDE4β separated two peaks of rolipram-sensitive PDE activity. The main peak eluted at a volume indicative of a ∼180 kDa protein but was preceded by a much larger form of the enzyme that had an estimated weight of 750 kDa. Size exclusion chromatography of CPPDE4α resolved a broad peak of activity with molecular weights spanning 50 to 200 kDa.
7. Of ten PDE inhibitors examined, none distinguished CPPDE4α from CPPDE4β with respect to their IC₅₀ values or their rank order of potency. RS-rolipram acted as a purely competitive inhibitor of cyclic AMP hydrolysis with K_is of 2 μM and 1.5 μM for CPPDE4α and CPPDE4β, respectively. In contrast to the membrane-associated enzyme(s), R-rolipram and nitraquazone were 4 to 19 fold less potent as inhibitors of CPPDE4α and CPPDE4β.
8. In intact macrophages, Ro 20-1724 and RS-rolipram potentiated isoprenaline-induced cyclic AMP accumulation under conditions where a PDE3 inhibitor, SK&F 94120, was essentially inactive.
9. These data demonstrate that the predominant cyclic AMP hydrolyzing activity in guinea-pig macrophages is a PDE4. Moreover, thermostability studies and size exclusion chromatography indicates the possible expression of two intrinsic, membrane-associated isoenzymes which can regulate the cyclic AMP content in intact cells. The finding that soluble and particulate forms of the same enzyme exhibit different sensitivities to rolipram and nitraquazone implies that PDE4 can change conformation. Finally, the identification of multiple molecular weight species of CPPDE4 suggests that this enzyme(s) might form multimeric complexes of variable association states.

     

Role of a Ca2+-Mg2+ ATPase on the mast cell surface in calcium transport and histamine secretion

We have previously reported the presence of an ATPase, stimulated by calcium and magnesium, on the outer surface of the rat peritoneal mast cell. Experiments in which the enzyme activity was enhanced or inhibited showed a relationship to histamine secretion. Enhanced enzyme activity with increasing concentrations of the substrate (ATP) was associated with a potentiation of histamine release, and a pronounced inhibition of the enzyme caused an inhibition of the release. In the present work we have studied the influx and efflux of calcium in mast cells in relation to the activity of the Ca2+-Mg2+ ATPase on the mast cell membrane. The enzyme activity is shown to be related to calcium influx and has no effect on calcium efflux. Stimulation of the enzyme with ATP is associated with increased calcium influx into the mast cell, and inhibition of the enzyme with AMP causes inhibition of the calcium uptake. In both cases calcium efflux is unaffected. The function of the enzyme is thus different from the calcium efflux enzyme on the cytoplasmic surface, described in other cells. In addition, the Ca2+-Mg2+ ATPase on the mast cell surface is neither stimulated by calmodulin nor inhibited by the calmodulin antagonists, trifluoperazine and W-7. In mast cells the low cytosolic calcium concentration seems to be maintained by Na+-Ca2+ countertransport. Phosphorylation of the Ca2+-Mg2+ ATPase on the mast cell is likely to be associated with Ca2+ release at the cytoplasmic surface of the plasma membrane. It is thus possible that ATP hydrolysis in the membrane stimulates the contraction of microfilaments in the membrane and the cytoskeleton, and promotes the migration of the granules to the plasma membrane.     

Inhibition in L-type Ca2+ channel by stimulation of protein kinase C in isolated guinea pig ventricular cardiomyocytes.

1. Electrophysiological effects of phorbol esters on the L-type Ca2+ current (ICa(L)) in isolated single ventricular cells from guinea pig hearts were investigated. 2. In whole-cell voltage-clamped myocytes, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) at 10(-7) M inhibited ICa(L). An antagonist of protein kinase C (PK-C), H-7, at 10(-5) M did not modify the TPA-induced inhibition. The time-course of inactivation process for ICa(L) was greatly slowed. 3. In cell-attached patch-clamp experiments, TPA (10(-7) M) also markedly decreased the opening of L-type Ca2+ channels. The conductance was unaffected. 4. Even H-7 (10(-5) M) alone inhibited the opening of the channels. Addition of TPA (10(-7)-10(-8) M) caused further decrease in the opening. 5. On the other hand, 4-alpha-phorbol-12,13-didecanoate (not a PK-C activator) had no effect on the Ca2+ channels. 6. These results indicate that the PK-C activation induced by TPA greatly depresses the opening of L-type Ca2+ channels in ventricular cell membranes.     

The effect of verapamil on the Ca2+-transporting and Ca2+-ATPase activity of isolated cardiac sarcolemmal preparations

1 Intestinal absorption and blood flow were determined in anaesthetized fed or fasted dogs following rapid intravenous injections of morphine (0.01, 0.1, 1 mg/kg). 2 3H2O and 22Na were used to determine the unidirectional fluxes of Na+ and H2O from saline perfused through the ileal lumen and the clearances of 3H2O were used to determine total and absorptive site blood flow. 3 Net Na+ and H2O absorption were increased at each dose of morphine in fed but not in fasted dogs, due primarily to increased absorptive fluxes. 4 Arterial pressure was decreased by morphine but mesenteric vein pressure was little affected. Absorptive site blood flow was increased by morphine due to decreased blood flow resistance but total blood flow resistance was little affected by morphine. 5 The absorptive fluxes of Na+ and H2O were correlated with absorpitve site blood flow in both fed and fasted animals. The secretory fluxes of Na+ and H2O were correlated with estimated capiliary pressure in fasted dogs but morphine decreased the the secretory fluxes at a given capillary pressure in dogs which had been fed. 6 Naloxone (0.12 mg, i.v.) reversed the effects of morphine. The effects of morphine on the gut were reversed more slowly than on systemic blood pressure. 7 It was concluded that morphine can increase net absorption in fed dogs by a selective increase in intestinal absorptive site blood flow and thus increase absorptive fluxes by a washout effect but that there is also an epithelial effect, sensitized by feeding, which reduces the secretory fluxes of Na+ and H2O.     

茶碱、咯利普兰和acetamide—45对GL62酵母细胞表达的人磷酸二酯酶4A的抑制作用

目的:研究GL62 酵母细胞中磷酸二酯酶4A(PDE4A)的诱导表达和茶碱、咯利普兰、N-对氟苄基-3-[(N-4-吡啶)-乙酰胺]-吲哚45(acetamide-45)对GL62酵母细胞诱导提取物中PDE4A活性的抑制作用.方法:克隆了人PDE4A基因的GL62酵母细胞在CuSO_4 150μmol/L条件下诱导表达PDE4A并提取,用高效液相色谱法测定PDE4A的活性.结果:GL62酵母细胞在CuSO_4诱导下的表达产物蛋白分子在62 kDa和83 kDa之间,表达产物的PDE4A的活性在3 h达到最大为(188±23)μmol·g~(-1)·min~(-1),其米氏常数 K_m为(17.7±2.6)μmol·L~(-1).茶碱,咯利普兰和acetamide-45对诱导提取物PDE4A活性的IC_(50)(95 ％可信限)分别为1642(989-2727),4.58(3.45-6.08)和275(170-444)μmol·L~(-1).结论:GL62酵母细胞经CuSO_4诱导可表达PDE4A,茶碱、咯利普兰和 acetamide-45能抑制其活性,GL62酵母细胞的诱导表达提取物可用来研究PDE4A及其抑制剂.     

Regulation of cAMP by phosphodiesterases in erythrocytes

The erythrocyte, a cell responsible for carrying and delivering oxygen in the body, has often been regarded as simply a vehicle for the circulation of hemoglobin. However, it has become evident that this cell also participates in the regulation of vascular caliber in the microcirculation via release of the potent vasodilator, adenosine triphosphate (ATP). The regulated release of ATP from erythrocytes occurs via a defined signaling pathway and requires increases in cyclic 3’,5’- adenosine monophosphate (cAMP). It is well recognized that cAMP is a critical second messenger in diverse signaling pathways. In all cells increases in cAMP are localized and regulated by the activity of phosphodiesterases (PDEs). In erythrocytes activation of either β adrenergic receptors (β₂AR) or the prostacyclin receptor (IPR) results in increases in cAMP and ATP release. Receptor-mediated increases in cAMP are tightly regulated by distinct PDEs associated with each signaling pathway as shown by the finding that selective inhibitors of the PDEs localized to each pathway potentiate both increases in cAMP and ATP release. Here we review the profile of PDEs identified in erythrocytes, their association with specific signaling pathways and their role in the regulation of ATP release from these cells. Understanding the contribution of PDEs to the control of ATP release from erythrocytes identifies this cell as a potential target for the development of drugs for the treatment of vascular disease.     

Stimulation of cardiac sarcolemmal (Na+--K+) ATPase activity by phosphorylase kinase.

Following preincubation with phosphorylase kinase, ATPase activities of heart sarcolemmal membranes were increased: total ATPase from 9.38+/-0.65 to 15.25+/-0.90 and ouabain-sensitive (Na+--K+)ATPase from 1.67+/-0.17 to 3.12+/-0.33 micron moles Pi/mg protein/h (mean +/- S.E. of 3 experiments); (Ca2+)ATPase and (Mg2+--Ca2+)-ATPase activities were not significantly altered due to phosphorylase kinase. Under these conditions, phosphorylase kinase catalyzed phosphorylation of sarcolemmal membranes. The kinase-catalyzed phosphorylation of membranes was increased by Ca2+ ions: at pH 6.8, 30% increase in phosphorylation was observed whereas at pH 8.5, 267% increase was noted due to this action. These findings support the view that Ca2+-dependent phosphorylation of membranes regulates (Na+--K+)ATPase.     

Antagonism by methylxanthines of purine nucleotide- and dipyridamole-induced inhibition of peristaltic activity of the guinea pig ileum

The effect of the methylxanthines (aminophylline, AMI and theophylline, THEO) on the responses to the purine pig ileum has been studied. ATP, ADP and adenosine produced concentration-dependent inhibition of peristaltic activity in non-treated, reserpine- or 6-hydroxydopamine-pretreated preparations; the relative potencies in descending order were ATP ? ADP > adenosine. This inhibition of peristaltic activity by the purine nucleotides was antagonized by low concentrations of the methylxanthines and this effect was reversible at all concentrations tested. The antagonism by THEO (5.0 μM) appeared competitive in nature. In contrast, peristaltic acdtivity inhibited by noradrenaline, isoprenaline, periarterial nerve stimulation (PNS) or papaverine was unaffected by the presence of the methylxanthines. Dipyridamole (5–10 μM) produced and inhibition in the late phase of the peristaltic activity, while lower concentrations (0.5–1.3 μM) potentiated the inhibitory response to ATP, ADP and adenosine without influencing the responses to noradrenaline, isoprenaline, PNS or papaverine. The methylxanthines selectively antagonized the inhivitory effects of dipyridamole alone or in combination with the purine nucleotides.It is proposed, that AMI and THEO exert a selective action on a specific ATP-receptor and that THEO may be a competitive antagonist of the purine nucleotides on peristaltic activity of the guinea pig ileum. Since 100-fold greater concentrations of AMI ot THEO are required to inhibit this preparation by inhibition of phosphodiesterase, it is highly unlikely that the inhibition of peristalsis by dipyridamole and the subsequent blockade by low concentrations of the methylxanthines involves the 3′, 5′-cyclic AMP pathway. Furthermore, the parallelism observed, between the effect of the methylxanthines on the inhibitory responses obtained with dipyridamole nad the effect of ATP, ADP and adenosine, supports the hypothesis that non-adrenergic, non-cholinergic inhibitory neurons releasing a purine nucleaotide as the neurotransmitter contribute to the descending inhibition observed during peristalsis.     

Effects of thiopental on Ca2+ currents and intracellular Ca2+ transient in single atrial cells from guinea pig

The effects of thiopental on Ca2+ currents and intracellular Ca2+ transient in single atrial cells from guinea pigs were studied by means of a whole-cell voltage-clamp method and Ca2+-sensitive fluorescent dye. Thiopental inhibited L-type voltage-dependent Ca2+ currents in a concentration-dependent manner (IC50=2.8.10(-5) mol/l). Moreover, the mode of Ca2+ current inhibition by thiopental showed no use dependency. Electrical stimulation-induced intracellular Ca2+ transient was significantly suppressed by 10(-5) mol/l thiopental. However, the caffeine-sensitive Ca2+ releasing pathway from sarcoplasmic reticulum was not affected by thiopental. Our results indicate that thiopental inhibits L-type Ca2+ currents, but not release of Ca2+ from sarcoplasmic reticulum. These results suggest that the negative inotropic action of thiopental is mainly due to inhibition of L-type Ca2+ channels in guinea pig atrial myocytes.     

Inhibition of alpha-receptor-induced Ca2+ release and Ca2+ influx by Mn2+ and La3+

The influence of Mn2+ and La3+ on alpha-receptor-stimulated Ca2+ movements was examined in arterial smooth muscle of the rabbit aorta. Both cations cause an inhibition of phenylephrine (PE) contractile response which exhibits a different pattern at low and high cation concentrations. At 0.1-1.0 mM inhibition by Mn2+ and La3+ was predominately due to a reduction in Ca2+ influx reflected as inhibition of the slow phase of contraction and reduction in PE-stimulated 45Ca uptake. PE log dose-response curves were shifted to the right in a non-parallel manner by 1 mM Mn2+ such that responses to lower PE concentrations were more inhibited. However, in the presence of 10 mM Mn2+ PE responses are equally inhibited at all PE levels. At 10 mM both Mn2+ and La3+ also inhibited PE-stimulated Ca2+ release resulting in a reduction in both the rapid phase of contraction and in the magnitude of PE stimulation of 45Ca efflux. The effects of Nm2+ (1 or 10 mM) on contraction and 45Ca efflux were rapidly reversible, while the effect of La3+ was not. Inhibition of Ca2+ release by 10 mM Mn2+ and La3+ was not caused by displacement of releasable Ca2+, but appeared to reflect their occupation of a superficially located receptor modulating site. The inhibition of Ca2+ influx by lower concentrations of Mn2+ may illustrate the functional consequence of configurational changes in the alpha 2-form of the receptor which have been recently described at lower concentrations of divalent cations.