The role of polyamines in beta-adrenergic stimulation of calcium influx and membrane transport in rat heart

The beta-adrenergic agonist 1-isoproterenol induced an early (less than 1 min) stimulation of endocytosis, amino acid transport and hexose transport, monitored by the temperature-sensitive uptake of horseradish peroxidase, alpha-aminoisobutyrate and 2-deoxyglucose, respectively, in rat ventricle cubes. This stimulation was time- and concentration-dependent and was maximum at 10(-8) M isoproterenol. The beta-adrenergic antagonist propranolol blocked isoproterenol stimulation of membrane transport, thereby confirming beta-adrenoceptor mediation; 2.5 mM EGTA, 1 mM LaCl2 and 100 microM verapamil blocked the hormonal response without affecting basal transport. The calcium ionophore A23187 caused an acute stimulation of endocytosis, hexose and amino acid transport. Isoproterenol rapidly (less than 30 s) stimulated 45Ca2+ influx. These data suggest that stimulus-response (stimulus-"transport") coupling is mediated by a rise in cytosolic Ca2+ concentration. A rapid (less than 30 to 60 s) increase in ornithine decarboxylase (ODC) activity, followed by an early (less than 1 to 2 min), sustained increase in putrescine, spermidine and spermine concentrations was evoked by 10(-7) M isoproterenol. The ODC inhibitor alpha-difluoromethylornithine (DFMO, 5 mM) suppressed the isoproterenol-induced increase in ODC and polyamine levels and the stimulation of 45Ca influx, endocytosis, hexose transport, and amino acid transport. Putrescine (0.5 mM) negated DFMO inhibition and restored the increase in polyamines, 45Ca influx, endocytosis, and transport of hexose and amino acid. These data suggest that polyamine synthesis is involved in isoproterenol stimulation of Ca2+ influx and membrane transport functions in ventricular myocardium. These findings are consistent with a model for signal transduction and stimulus-response coupling in which polyamines function as intracellular messengers to generate cytosolic Ca2+ signals by stimulating Ca2+ influx.     

Beta-adrenergic stimulation of Ca2+ fluxes, endocytosis, hexose transport, and amino acid transport in mouse kidney cortex is mediated by polyamine synthesis.

We recently found that the beta-adrenergic agonist 1-isoproterenol evokes a rapid (less than 5 min) Ca2+- and receptor-dependent stimulation of endocytosis, hexose transport, and amino acid transport in mouse renal cortex involving proximal tubule cells. This response is associated with increased Ca2+ fluxes and a mobilization of mitochondrial calcium, suggesting that stimulus-response (stimulus-"transport") coupling is mediated by cytosolic Ca2+. We show here that 1 microM isoproterenol evokes a rapid (less than 60 sec) transient increase in the activity of ornithine decarboxylase followed by an early (less than 2 min) sustained increase in putrescine, spermidine, and spermine concentrations in mouse kidney cortex slices in vitro. Small doses of isoproterenol (down to 24 nmol/kg) elicited a rapid (less than 2 min) increase in polyamines in vivo. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine (5 mM) suppressed the testosterone-induced increase in polyamine levels and rates of endocytosis, hexose transport, and amino acid transport, measured by horseradish peroxidase, [14C]aminoisobutyric acid, and deoxy[3H]glucose uptake. alpha-Difluoromethylornithine also blocked the isoproterenol-induced increase in 45Ca influx and efflux and 45Ca redistribution; 0.5 mM putrescine nullified alpha-difluoromethylornithine inhibition and restored the increment in polyamines, 45Ca fluxes, endocytosis, hexose transport, and amino acid transport. These data implicate polyamine synthesis in isoproterenol stimulation of Ca2+ fluxes and membrane transport processes and support a model for signal transduction and stimulus-response coupling in which ornithine decarboxylase activation and polyamine synthesis play a pivotal role in regulating Ca2+ fluxes. In this model the polyamines generate local Ca2+ signals by stimulating Ca2+ influx or mobilizing intracellular calcium (or both) through a cation exchange reaction.     

Phorbol ester and dioctanoylglycerol stimulate membrane association of protein kinase C and have a negative inotropic effect mediated by changes in cytosolic Ca2+ in adult rat cardiac myocytes

We used left ventricular myocytes from adult rats to investigate the effect of 4 beta-phorbol 12-myristate 13-acetate (PMA) and of sn-1,2-dioctanoylglycerol (DiC-8) on the membrane association of protein kinase C (PKC), cytosolic [Ca2+], (Cai) homeostasis, and the contractile properties of single cardiac cells. Because PKC activity is known to be highly Ca2+ sensitive, the K+ concentration of the bathing medium was raised from 5 to 30 mM in some experiments, a perturbation known to depolarize the cell and increase Cai. In cell suspensions both PMA (3 x 10(-10) and 3 x 10(-7) M) and DiC-8 (10(-5) and 10(-4) M) increased membrane association of PKC. The effect of PMA (10(-7) M) on PKC translocation was enhanced in 30 mM KCl compared with 5 mM KCl. During steady field stimulation at 1 Hz in 1 mM bathing [Ca2+], both PMA (10(-7) M) and DiC-8 (10(-5) M) decreased twitch amplitude to approximately 60% of control in 5 mM KCl, and the negative inotropic effect of either drug was more pronounced in 30 mM KCl than in 5 mM KCl. In single cardiac myocytes loaded with the Ca2+ indicator indo-1 and bathed in 5 mM KCl, we simultaneously measured cell length and Cai. The myofilament responsiveness to Ca2+ was assessed by the relation between contraction amplitude and the peak of the Cai transient. The negative inotropic effect of both PMA and DiC-8 was related to a diminished amplitude of the Cai transient and not to a decreased myofilament responsiveness to Ca2+. In the absence of electrical stimulation, PMA (10(-7) M) and DiC-8 (10(-5) M) decreased the frequency of contractile waves due to spontaneous Ca2+ release from the sarcoplasmic reticulum, and DiC-8 also decreased resting Cai. Thus, activation of PKC, which is thought to occur as part of the response of cardiac muscle to alpha 1-adrenergic stimulation, is associated with a negative inotropic action due to a smaller Cai transient rather than to a decrease in the myofilament responsiveness to Ca2+. These effects on the membrane association of PKC and on contractility are enhanced by cell depolarization achieved by raising [KCl] in the bathing medium.     

Kappa and delta opioid receptor stimulation affects cardiac myocyte function and Ca2+ release from an intracellular pool in myocytes and neurons.

We investigated the effects of mu, delta, and kappa opioid receptor stimulation on the contractile properties and cytosolic Ca2+ (Cai) of adult rat left ventricular myocytes. Cells were field-stimulated at 1 Hz in 1.5 mM bathing Ca2+ at 23 degrees C. The mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (10(-5) M) had no effect on the twitch. The delta-agonists methionine enkephalin and leucine enkephalin (10(-10) to 10(-6) M) and the kappa-agonist (trans-(dl)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclo-hexyl]- benzeneacetamide)methanesulfonate hydrate (U-50,488H; 10(-7) to 2 x 10(-5) M) had a concentration-dependent negative inotropic action. The sustained decrease in twitch amplitude due to U-50,488H was preceded by a transient increase in contraction. The effects of delta- and kappa-receptor stimulation were antagonized by naloxone and (-)-N-(3-furyl-methyl)-alpha-normetazocine methanesulfonate, respectively. In myocytes loaded with the Ca2+ probe indo-1, the effects of leucine enkephalin (10(-8) M) and U-50,488H (10(-5) M) on the twitch were associated with similar directional changes in the Cai transient. Myofilament responsiveness to Ca2+ was assessed by the relation between twitch amplitude and systolic indo-1 transient. Leucine enkephalin (10(-8) M) had no effect, whereas U-50,488H (10(-5) M) increased myofilament responsiveness to Ca2+. We subsequently tested the hypothesis that delta and kappa opioid receptor stimulation may cause sarcoplasmic reticulum Ca2+ depletion. The sarcoplasmic reticulum Ca2+ content in myocytes and in a caffeine-sensitive intracellular Ca2+ store in neurons was probed in the absence of electrical stimulation via the rapid addition of a high concentration of caffeine from a patch pipette above the cell. U-50,488H and leucine enkephalin slowly increased Cai or caused Cai oscillations and eventually abolished the caffeine-triggered Cai transient. These effects occurred in both myocytes and neuroblastoma-2a cells. In cardiac myocyte suspensions U-50,488H and leucine enkephalin both caused a rapid and sustained increase in inositol 1,4,5-trisphosphate. Thus, delta and kappa but not mu opioids have a negative inotropic action due to a decreased Cai transient. The decreased twitch amplitude due to kappa-receptor stimulation is preceded by a transient increase in contractility, and it occurs despite an enhanced myofilament responsiveness to Ca2+. The effects of delta and kappa opioids appear coupled to phosphatidylinositol turnover and, at least in part, may be due to sarcoplasmic reticulum Ca2+ depletion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+ dependence of alpha-adrenergic effects on the contractile properties and Ca2+ homeostasis of cardiac myocytes.

alpha-Adrenergic stimulation is known to enhance myocardial contractility. Adult rat left ventricular myocytes bathed in 1 mM [Ca2+] (Ca0) and electrically stimulated at 0.2 Hz responded to alpha-adrenergic stimulation with 50 microM phenylephrine and 1 microM propranolol with an increase in twitch amplitude to 177.1 +/- 25.6% of control (mean +/- SEM). In contrast, when cell Ca2+ loading was increased by bathing cells in 5 mM Ca0, alpha-adrenergic stimulation decreased twitch amplitude to 68.6 +/- 8.2% of control. Time-averaged cytosolic [Ca2+] of cells in 1.0 mM Ca0 is enhanced via an increase in the frequency of electrical stimulation. When myocytes were stimulated at 2 Hz in 1 mM Ca0, alpha-adrenergic stimulation did not increase twitch amplitude (103.8 +/- 12.4% of control). In myocytes loaded with the Ca2+ probe into-1, alpha-adrenergic effects during stimulation at 0.2 Hz (an increase in twitch amplitude in 1 mM Ca0 and a decrease in twitch amplitude in 5 mM Ca0) were associated with similar changes in the indo-1 transient. In 5 mM Ca0, spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) occurred in the diastolic interval between twitches (2.9 +/- 1.4 spontaneous SR Ca2+ oscillations/min; n = 7); alpha-adrenergic stimulation abolished these oscillations in six of seven cells. Thus, an increase in the frequency of spontaneous diastolic SR Ca2+ release (i.e., Ca2+ overload) is not the mechanism for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. In experiments with unstimulated myocytes, we determined whether the effect of alpha-adrenergic stimulation on cell Ca2+ homeostasis and oscillatory SR Ca2+ release observed in 5 mM Ca0 occurs only during electrical stimulation, when voltage-dependent currents are operative, or also at rest. Unstimulated rat ventricular myocytes in 5 mM Cao exhibit oscillatory SR Ca2+ release; alpha-adrenergic stimulation decreased the frequency of these oscillations to 53.9 +/- 8.9% of control, and this effect was blocked by 1 microM prazosin. In unstimulated indo-1-loaded myocytes alpha-adrenergic stimulation decreased the resting indo-1 fluorescence ratio in 5 mM Ca0, whereas it had no effect in 1 mM Ca0. Additional experiments were aimed at defining a role for Ca(2+)-activated, phospholipid-dependent protein kinase C (PKC) for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. Short-term preexposure to 0.1 microM 4 beta-phrobol 12-myristate 13-acetate (PMA) has been shown to maximally activate PKC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Failure of ornithine decarboxylase inhibition to alter small intestinal epithelial repair after transient segmental ischaemia.

To evaluate the roles of ornithine decarboxylase (ODC) and polyamines in the regulation of epithelial repair, rabbit mid-small intestine after transient ischaemic villus injury in the presence and absence of DL-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC was studied. Rabbits received 2% (w/v) DFMO in drinking water for two days before undergoing a sham laparotomy, or a 90 minute mesenteric vascular occlusion of 20 cm of mid-intestine. DFMO fed and control rabbits were studied four, 24, 72, or 120 hours after this ischaemic intestinal injury. In controls, ischaemic injury caused shortened villi at four hours (p less than 0.01), diminished mucosal sucrase and alkaline phosphatase activities at 24 hours (p less than 0.05), but raised ODC (p less than 0.001) and thymidine kinase (p less than 0.01) activities at four hours with recovery by 72 hours. DFMO treatment significantly reduced ODC activity at all stages of the experiment and significantly inhibited the rise in activity observed after injury (p less than 0.01). Mucosal concentrations of the polyamines, spermidine and spermine, were similar in the sham operated groups; four hours and 24 hours after ischaemia, they increased in the DFMO animals (p less than 0.01) but fell (p less than 0.05) in those that did not receive DFMO. After ischaemic injury, DFMO treatment inhibited ODC but failed to influence recovery of villus structure or enzyme activities in the small intestine. We conclude that ODC and the polyamines, spermidine and spermine, are not key regulators of small intestinal repair after transient ischaemia.     

Calcium entry via Na/Ca exchange during the action potential directly contributes to contraction of failing human ventricular myocytes

Prolongation of the Ca2+ transient and action potential (AP) durations are two characteristic changes in myocyte physiology in the failing human heart. The hypothesis of this study is that Ca2+ influx via reverse mode Na+/Ca2+ exchanger (NCX) or via L-type Ca2+ channels directly activates contraction in failing human myocytes while in normal myocytes this Ca2+ is transported into the sarcoplasmic reticulum (SR) to regulate SR Ca2+ stores. METHODS: Myocytes were isolated from failing human (n=6), nonfailing human (n=3) and normal feline hearts (n=9) and whole cell current and voltage clamp techniques were used to evoke and increase the duration of APs (0.5 Hz, 37 degrees C). Cyclopiazonic acid (CPA 10(-6) M), nifedipine (NIF;10(-6) M) and KB-R 7943 (KB-R; 3x10(-6) M) were used to reduce SR Ca2+ uptake, Ca2+ influx via the L-type Ca2+ current and reverse mode NCX, respectively. [Na+)i was changed by dialyzing myocytes with 0, 10 and 20 mM Na(+) pipette solutions. RESULTS: Prolongation of the AP duration caused an immediate prolongation of contraction and Ca2+ transient durations in failing myocytes. The first beat after the prolonged AP was potentiated by 21+/-5 and 27+/-5% in nonfailing human and normal feline myocytes, respectively (P<0.05), but there was no significant effect in failing human myocytes (+5+/-4% vs. steady state). CPA blunted the potentiation of the first beat after AP prolongation in normal feline and nonfailing human myocytes, mimicking the failing phenotype. NIF reduced steady state contraction in feline myocytes but the potentiation of the first beat after AP prolongation was unaltered (21+/-3% vs. base, P<0.05). KB-R reduced basal contractility and abolished the potentiation of the first beat after AP prolongation (2+/-1% vs. steady state). Increasing [Na+]i shortened AP, Ca2+ transient and contraction durations and increased steady state and post AP prolongation contractions. Dialysis with 0 Na+ eliminated these effects. CONCLUSIONS: Ca2+ enters both normal and failing cardiac myocytes during the late portion of the AP plateau via reverse mode NCX. In (normal) myocytes with good SR function, this Ca(2+) influx helps maintain and regulate SR Ca2+ load. In (failing) human myocytes with poor SR function this Ca2+ influx directly contributes to contraction. These studies suggest that the Ca2+ transient of the failing human ventricular myocytes has a higher than normal reliance on Ca2+ influx via the reverse mode of the NCX during the terminal phases of the AP.     

Effects of ryanodine and caffeine on contractility, membrane voltage, and calcium exchange in cultured heart cells

To investigate the mechanisms of action of ryanodine and caffeine, changes in mechanical and electrical activity caused by these agents were correlated with alterations in 45Ca fluxes and cell Ca contents in chick embryo ventricular cell monolayer cultures. Ryanodine (10(-10)-10(-5) M) irreversibly decreased contraction amplitude by 10-70% relative to control in a concentration-dependent manner with minimal effects on electrical activity. Ryanodine caused a slight decrease in rapid 45Ca uptake, but no change in total exchangeable calcium content or rapid 45Ca efflux. Caffeine (1-20 mM) caused a transient (less than 10 seconds) 5-12% increase in contraction amplitude followed by a sustained 9-76% decrease in contraction amplitude and a 10 mV decrease in diastolic membrane voltage. Caffeine caused a decrease in rapid 45Ca uptake, a decrease in total exchangeable calcium content, and an increase in rapid 45Ca efflux. These results suggest that caffeine produces a decrease in sarcoplasmic reticulum (SR) Ca2+ uptake, and/or an increase in SR Ca2+ release that eventually depletes the SR of Ca2+, presumably accounting for the negative inotropic effect. The ryanodine effects on contraction are more difficult to account for solely in terms of alterations of transsarcolemmal Ca2+ fluxes and Ca2+ contents. Our data indicate an important role for the SR in excitation-contraction coupling in cultured chick embryo ventricular cells and suggest that SR Ca2+ is part of the rapidly exchanging Ca2+ compartment noted in 45Ca flux studies.     

Influence of cell isolation and incubation procedures on Ca2+ dependence of glucose transport in isolated cardiac myocytes

Insulin stimulates hexose transport in isolated myocytes of adult rats but data are conflicting regarding the Ca2+-dependence of this effect, demonstrated previously in intact heart muscle. 3-O-methyl-D-glucose and 2-deoxy-D-glucose transport was compared in cells prepared and incubated by different published procedures. Ca2+-dependence of hexose transport stimulation by insulin was found only with incubation in a modified Joklik tissue culture medium buff red with bicarbonate at pH 7.4. At pH 7.0 or when bicarbonate was replaced by N-2-hydroxyethylpiperazine-N'-2 ethane sulfonic acid (HEPES) stimulation by insulin was not Ca2+-dependent, nor was there a Ca2+-dependent increment in insulin effect in physiological saline media regardless of pH or buffer (HEPES, 3-[N-morpholino] propanesulfonic acid (MOPS) or bicarbonate). The ATP content of cells incubated in HEPES or MOPS media was reduced. Ca2+-dependence seems to require the presence of bicarbonate and another factor(s) in tissue culture medium. The response is also influenced by the isolation procedure and may be related in part to preservation of an intact glycocalix. Thus, details in cell isolation and incubation procedures may strongly influence the behaviour of the cells. Isolated cardiac myocytes are acceptable as a valid model only if it can be demonstrated in each specific case that the characteristics present in intact tissue are maintained unaltered in the isolated cells.     

Ethanol inhibits electrically-induced calcium transients in isolated rat cardiac myocytes

The fluorescent Ca2+ indicator fura-2 was used to follow cytosolic Ca2+ transients during excitation-contraction coupling in suspensions of isolated rat heart cells induced to beat synchronously by electrical field stimulation. The Ca2+ transient reached a maximum at about 30 ms after application of the electrical stimulus and then relaxed to the basal level over the following 200 ms. Treatment of the myocytes with 0.25 to 2.0% ethanol (40 to 340 mM) caused a decrease in the peak of the Ca2+ transient, with no apparent change in the time to peak. This effect of ethanol occurred progressively over a period of about 1 min before a new stable state was achieved. At 1% ethanol the peak Ca2+ level was reduced by 50%. Ethanol reversed the stimulatory effect of isoproterenol on peak Ca2+ and at high levels of ethanol the beta-adrenergic agonist no longer caused any enhancement of the Ca2+ transient. Ethanol did not cause any marked change in the basal Ca2+ level between beats. The effects of ethanol were readily reversible. These results suggest that the negative inotropic effect of ethanol observed in intact cardiac muscle preparations may result in part from interference with the Ca2+ fluxes responsible for excitation-contraction coupling in ventricular myocytes.     

Sodium-calcium exchange initiated by the Ca2+ transient: an arrhythmia trigger within pulmonary veins.

OBJECTIVES: The hypothesis that an increased or prolonged Ca2+ transient during an abbreviated action potential can give rise to early afterdepolarizations (EADs) and triggered arrhythmia by enhanced forward sodium-calcium (Na-Ca) exchange was examined. BACKGROUND: Because pulmonary veins have the shortest action potential of any cardiac tissue, we examined this hypothesis in canine pulmonary vein sleeves during interventions further shortening the action potential and increasing the calcium transient. METHODS: Extracellular bipolar electrode, intracellular microelectrode, and isometric force (a surrogate marker for the Ca2+ transient) recordings were obtained from superfused canine pulmonary veins. RESULTS: An elevation and prolongation of the terminal phase of repolarization (EADs) were observed during interventions increasing contractile force; isoproterenol or norepinephrine (3.2 x 10(-11) to 3.2 x 10(-7)M), hypothermia, and pacing (post-extrasystolic potentiation, post-pacing pause). The EAD formation was prevented by ryanodine (10 microM) or reversed by transiently increasing [Ca2+](o) from 1.35 to 5 mM (inhibition of forward Na-Ca exchange). Pacing-induced EADs were enhanced by re-introduction of normal Tyrode solution (Na+ = 130 mM) after substitution of 30 mM NaCl with 30 mM LiCl (stimulation of forward Na-Ca exchange). With norepinephrine or isoproterenol (3.2 x 10(-8)M) + acetylcholine (10(-7)M) (to enhance the Ca2+ transient and further shorten the abbreviated action potential, respectively), tachycardia-pause initiated arrhythmia (1,132 +/- 153 beats/min) lasting >1 s was observed. Rapid firing was prevented by either suppression of the Ca2+ transient (ryanodine) or transiently increasing [Ca2+](o). CONCLUSIONS: The data show EAD formation in superfused canine pulmonary veins, enhanced by an increased Ca2+ transient and increased Na-Ca exchange current. With subsequent shortening of the action potential with acetylcholine, tachycardia-pause triggers rapid firing within the PV sleeve.     

Testosterone enhances calcium reabsorption by the kidney

The kidney is a target tissue for androgens, but the role of these hormones in the regulation of calcium (Ca2+) reabsorption remains unclear. The present study examines the effects of testosterone on Ca2+ transport by the luminal membranes of proximal and distal nephrons of the rabbit kidney. Tubule suspensions were pre-incubated in the presence or absence of the hormone, and 45C2+ uptake by the luminal membranes was measured using the rapid filtration technique. In the proximal tubules, testosterone did not influence Ca2+ uptake. In the distal tubules, a 5 min incubation with the hormone increased this uptake with a maximal response at 10(-10)M. Ca2+ transport by the distal membranes shows a dual kinetics. Testosterone enhanced the Vmax value of the low affinity component. In an attempt to identify the underlying mechanisms involved in this action, several messenger inhibitors were introduced in the tubule suspension. PD 98059 and U0 126 as well as AG 99 and genistein interfered with the hormone action suggesting the implication of a MEK kinase and a tyrosine kinase. To determine the type of the channels involved in this effect, Ca2+ uptake was measured in the presence of diltiazem, omega-conotoxin MVIIC and mibefradil, i.e. selective inhibitors of the L-type, P/Q type and T-type channels. An inhibition of Ca2+ transport was observed exclusively with mibefradil. These results indicate that testosterone enhances Ca2+ transport by opening a T-type Ca2+ channel in the distal luminal membrane, via MEK kinase and tyrosine kinase dependent mechanisms.     

Bimodal effect of stimulation on light fluctuation transients monitoring spontaneous sarcoplasmic reticulum calcium release in rat cardiac muscle

Microscopic, myofilament motion caused by spontaneous oscillatory Ca2+ release from the sarcoplasmic reticulum (SR) of unstimulated rat papillary muscles produces scattered light intensity fluctuations (SLIF) in a laser beam scattered by the tissue. SLIF frequency increases with Ca2+ loading of resting muscle. We used novel time-gated SLIF measurements to determine how electrical stimulation (which per se both induces SR Ca2+ release and modulates total cellular Ca2+ loading) affects SLIF. Stimulation of thin rat, right ventricular muscles at 1 Hz in bathing [Ca2+] (Ca0) of 1.5 mM at 29 degrees C abolished SLIF for 5-7 seconds; SLIF then reappeared and monotonically increased for 10-15 seconds to reach the steady resting level. Resting force transients paralleled those of SLIF. The magnitude of depression and time course of recovery of both resting force and SLIF at this Ca0 vary inversely with the rate of prior stimulation and the number of stimuli given. An increase in Ca0 or disablement of the Na-K pump increased both resting SLIF and force; transient stimulation under these conditions (i.e., in a 2.5-5.5-second "diastolic window" after cessation of stimulation) augmented SLIF and force above the resting level. Isoproterenol caused a modest reduction of resting SLIF, but it transiently increased SLIF after stimulation up to 10-fold above the resting level. Nifedipine did not affect resting SLIF but transiently depressed SLIF after stimulation. Ryanodine abolished SLIF both after stimulation and at rest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of polyamine synthesis by alpha-difluoromethylornithine and its effects on pancreatic secretion and growth in the rat

The role played by the polyamines in mediating the pancreatic growth and secretory responses to hormonal stimulation is uncertain. The effect of an inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethylornithine (DFMO), on rat pancreatic protein secretion and synthesis and on growth in response to hormonal stimulation was therefore studied. Anesthetized rats were given an intravenous injection of DFMO (50, 100, or 150 mg/kg), followed by a 7-h continuous infusion (15, 25, or 35 mg/kg/h, respectively). After a basal 1-h period an intravenous infusion of 2.5 micrograms/kg/h of the cholecystokinin-like peptide Thr28Nle31CCK25-33 (CCK-LP) was added and continued for 6 h. The control rats received CCK-LP only. The ODC activity in the pancreas was markedly reduced by DFMO, but DFMO did not affect pancreatic juice volume or protein output. In another series conscious rats were given a continuous intravenous infusion of 2.5 micrograms/kg/h of CCK-LP for 8, 24, and 48 h or 5.0 micrograms/kg/h of secretin for 8 and 48 h, with or without DFMO (100 mg/kg as an injection initially and thereafter 25 mg/kg/h). The ODC activity and putrescine concentration in the pancreas were significantly reduced by DFMO at 8 and 24 h but not at 48 h. DFMO also significantly reduced the activities of RNA polymerase, DNA polymerase, and thymidine kinase at 24 h, but not at 48 h. The present study thus indicates that polyamines play a role in the initiation of the growth response to hormonal stimulation but does not support a similar dependence for early pancreatic protein synthetic and secretory responses.     

Inhibition of arginine vasopressin-stimulated Na+ K+ ATPase activity by difluoromethyl ornithine in the rat renal medulla

Arginine vasopressin (AVP) stimulates Na+ K+ ATPase and ornithine decarboxylase (ODC) activity in the rat medullary thick ascending limb. The effect of difluoromethyl ornithine (DFMO), a specific inhibitor of ODC activity, on AVP-stimulated Na+ K+ ATPase activity was evaluated using a cytochemical bioassay. Peaks in Na+ K+ ATPase activity in cultured rat renal segments which occurred after tissue had been exposed to 1 fmol AVP/l were completely inhibited by the addition of 20 mmol DFMO/l to the culture medium containing AVP. The addition of 20 mmol DFMO/l to the culture medium containing AVP in the concentration range 0.001-10 fmol/l inhibited completely the stimulation of Na+ K+ ATPase activity by AVP. The response of Na+ K+ ATPase to increasing doses of ATP (10-40 g polypeptide/l) was not influenced by the addition of 20 mmol DFMO/l to the culture medium containing AVP, suggesting that the prevention of AVP-stimulated Na+ K+ ATPase activity by DFMO was not due to a direct effect on the enzyme.     

A new, nongenomic estrogen action: the rapid release of intracellular calcium.

We have investigated the effects of steroids on the intracellular calcium ion concentration [Ca2+]i in chicken granulosa cells obtained from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 100 +/- 5 nM. There was an immediate (i.e. less than 5 sec) 4- to 8-fold increase in [Ca2+]i in all of the 76 cells examined after the addition of 10(-7) M estradiol-17 bdta. Estradiol-17 beta was effective between 10(-10)-10(-6) M. Estradiol-17 alpha, estrone, and estriol (10(-8)-10(-6) M) were as effective as estradiol-17 beta, but the progestins, pregnenolone, and progesterone, and the androgens, testosterone, androstenedione, or 5 alpha-dihydrotestosterone were ineffective at concentrations up to 10(-5) M. The prompt estradiol-17 beta-induced [Ca2+]i spike was not affected by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating them with the Ca2+ channel blockers lanthanum (1 mM), cobalt (5 mM), methoxyverapamil (D600; 50 microM), or nifedipine (20 microM). The estrogen-triggered [Ca2+]i surge was also not affected by pretreating the cells with the conventional estrogen receptor antagonist tamoxifen (10(-5) M), or the RNA and protein synthesis inhibitors actinomycin D (1 microgram/ml) and cycloheximide (1 microgram/ml), but was abolished by pretreating the cells with inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) and U-73,122 (2.5 microM). The closely related, but inactive, compound U-73,343 (1 microM) did not affect the estrogen-triggered [Ca2+]i surge. Estradiol-17 beta (10(-7) M), but not progesterone (10(-5) M), also triggered a large [Ca2+]i surge in pig granulosa cells, which, like the [Ca2+]i surge in chicken granulosa cells, was almost immediate, transient, and unaffected by incubation in Ca(2+)-free medium or pretreatment with methoxyverapamil (D600; 50 microM), lanthanum (1 mM), or tamoxifen (10(-5)M). However, granulosa cells from immature rats primed with diethylstilbestrol or PMSG did not respond to estradiol-17 beta, even at concentrations as high as 10(-5) M, although they promptly generated a [Ca2+]i transient upon exposure to LHRH (10(-5) M). These results suggest that estrogens almost instantaneously trigger the release of Ca2+ from intracellular stores which may be mediated through phosphoinositide breakdown. The striking rapidity of this estrogen-induced internal Ca2+ mobilization is consistent with the activation of a cell surface receptor which is different from the conventional slowly acting, gene-stimulating nuclear estrogen receptor.     

Calcium control of growth hormone release from chicken pituitary glands in vitro

The influence of calcium on the basal and stimulated release of growth hormone (GH) from chicken pituitary glands has been determined in vitro. Basal GH release occurred in Ca2+ deficient media, although it was increased in proportion to the medium Ca2+ concentration. Growth hormone release was stimulated by 10(-7)-10(-9) M thyrotrophin-releasing hormone (TRH), maximal stimulation being observed in the presence of 10(-8) M TRH and 1.5 mM Ca2+. Decreases in the Ca2+ concentration (to 0.75, 0.375, or 0 mM) suppressed the GH response to 10(-8) M TRH, as did increases (to 3.0 and 6.0 mM) in the Ca2+ concentration. These results suggest that GH release in chickens is regulated by Ca2+-dependent mechanisms.     

Influence of interleukin-2 on Ca2+ handling in rat ventricular myocytes

In the present study, we examined the effect of interleukin-2 (IL-2) on cardiomyocyte Ca(2+) handling. The effects of steady-state and transient changes in stimulation frequency on the intracellular Ca(2+) transient were investigated in isolated ventricular myocytes by spectrofluorometry. In the steady state (0.2 Hz) IL-2 (200 U/ml) decreased the amplitude of Ca(2+) transients induced by electrical stimulation and caffeine. At 1.25 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), when the stimulation frequency increased from 0.2 to 1.0 Hz, diastolic Ca(2+) level and peak intracellular Ca(2+) concentration ([Ca(2+)](i)), as well as the amplitude of the transient, increased. The positive frequency relationships of the peak and amplitude of [Ca(2+)](i) transients were blunted in the IL-2-treated myocytes. The effect of IL-2 on the electrically induced [Ca(2+)](i) transient was not normalized by increasing [Ca(2+)](o) to 2.5 mM. IL-2 inhibited the frequency relationship of caffeine-induced Ca(2+) release. Blockade of sarcoplasmic reticulum (SR) Ca(2+)-ATPase with thapsigargin resulted in a significant reduction of the amplitude-frequency relationship of the transient similar to that induced by IL-2. The restitutions were not different between control and IL-2 groups at 1.25 mM [Ca(2+)](o), which was slowed in IL-2-treated myocytes when [Ca(2+)](o) was increased to 2.5 mM. There was no difference in the recirculation fraction (RF) between control and IL-2-treated myocytes at both 1.25 and 2.5 mM [Ca(2+)](o). The effects of IL-2 on frequency relationship, restitution, and RF may be due to depressed SR functions and an increased Na(+)-Ca(2+) exchange activity, but not to any change in L-type Ca(2+) channels.     

Action potential prolongation in cardiac myocytes of old rats is an adaptation to sustain youthful intracellular Ca2+ regulation

Advanced age in rats is accompanied by reduced expression of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA-2). The amplitudes of intracellular Ca2+ (Ca2+(i)) transients and contractions in ventricular myocytes isolated from old (23-24-months) rats (OR), however, are similar to those of young (4-6-months) rat myocytes (YR). OR myocytes also manifest slowed inactivation of L-type Ca2+ current (I(CaL)) and marked prolongation of action potential (AP) duration. To determine whether and how age-associated AP prolongation preserves the Ca2+(i) transient amplitude in OR myocytes, we employed an AP-clamp technique with simultaneous measurements of I(CaL) (with Na+ current, K+ currents and Ca2+ influx via sarcolemmal Na+-Ca2+ exchanger blocked) and Ca2+(i) transients in OR rat ventricular myocytes dialyzed with the fluorescent Ca2+ probe, indo-1. Myocytes were stimulated with AP-shaped voltage clamp waveforms approximating the configuration of prolonged, i.e. the native, AP of OR cells (AP-L), or with short AP waveforms (AP-S), typical of YR myocytes. Changes in SR Ca2+ load were assessed by rapid, complete SR Ca2+ depletions with caffeine. As expected, during stimulation with AP-S vs AP-L, peak I(CaL) increased, by 21+/-4%, while the I(CaL) integral decreased, by 19+/-3% (P<0.01 for each). Compared to AP-L, stimulation of OR myocytes with AP-S reduced the amplitudes of the Ca2+(i) transient by 31+/-6%, its maximal rate of rise (+dCa2+(i)/dt(max); a sensitive index of SR Ca2+ release flux) by 37+/-4%, and decreased the SR Ca2+ load by 29+/-4% (P<0.01 for each). Intriguingly, AP-S also reduced the maximal rate of the Ca2+(i) transient relaxation and prolonged its time to 50% decline, by 35+/-5% and 33+/-7%, respectively (P<0.01 for each). During stimulation with AP-S, the gain of Ca2+-induced Ca2+ release (CICR), indexed by +dCa2+(i)/dt(max)/I(CaL), was reduced by 46+/-4% vs AP-L (P<0.01). We conclude that the effects of an application of a shorter AP to OR myocytes to reduce +dCa2+(i)/dt(max) and the Ca2+ transient amplitude are attributable to a reduction in SR Ca2+ load, presumably due to a reduced I(CaL) integral and likely also to an increased Ca2+ extrusion via sarcolemmal Na+-Ca2+ exchanger. The decrease in the Ca2+(i) transient relaxation rate in OR cells stimulated with shorter APs may reflect a reduction of Ca2+/calmodulin-kinase II-regulated modulation of Ca2+ uptake via SERCA-2, consequent to a reduced local Ca2+ release in the vicinity of SERCA-2, also attributable to reduced SR Ca2+ load. Thus, the reduction of CICR gain during stimulation with AP-S is the net result of both a diminished SR Ca2+ release and an increased peak I(CaL). These results suggest that ventricular myocytes of old rats utilize AP prolongation to preserve an optimal SR Ca2+ loading, CICR gain and relaxation of Ca2+(i) transients.     

Calcium homeostasis in rabbit ventricular myocytes. Disruption by hypochlorous acid and restoration by dithiothreitol.

Hypochlorous acid (HOCl) is a toxic oxidant produced by neutrophils at sites of cardiac inflammation. To examine the effect of this oxidant on Ca2+ homeostasis in the heart, isolated rabbit ventricular myocytes were iontophoretically loaded with the Ca2+ indicator fura 2 and superfused with 100 microM HOCl under voltage-clamp conditions. Ca2+ transients and the corresponding Ca2+ currents were elicited by 300-msec depolarizing pulses from -40 to 0 mV. Within 200 seconds after HOCl addition, the amplitude of the Ca2+ transients was reduced from 402 +/- 89 to 82 +/- 29 nM (p less than 0.01) while intracellular free ([Ca2+]i increased from 78 +/- 16 to 265 +/- 48 nM (p less than 0.01). During this time, the amplitude of the slow inward currents increased by 10%, while steady-state holding current remained stable. This sustained steady-state rise in [Ca2+]i occurred even in the absence of extracellular Ca2+ but was virtually abolished by a 20-second preexposure to 10 mM caffeine, suggesting that the major source of this Ca2+ was the sarcoplasmic reticulum. Although washout of HOCl failed to induce recovery, subsequent exposure to the dithiol reducing agent dithiothreitol caused a rapid restoration of both the steady-state [Ca2+]i and Ca2+ transient amplitude. We conclude that 1) HOCl caused a rise of [Ca2+]i by inducing the release of Ca2+ from internal stores and impairing cellular extrusion mechanisms and 2) these effects occur through alteration of protein thiol redox status.