SUMMARY1. Calcium current (Ica) was recorded in single rat heart cells at two periods during development: (1) at 2-7 days post-partum (neonatal), and (2) at 6-8 weeks (adult).2. We measured both transient and steady-state components of ICa and could describe ICa in terms of the steady-state activation (d.) and inactivation (fV.) parameters, the channel reversal potential (Echannel) and a relative conductance parameter, gr -3. In adult single cells, the application of ryanodine (10 ,gM), an agent known to alter the function of the sarcoplasmic reticulum (SR), abolished contraction rapidly and increased ICa' Ryanodine also produced a 13 mV shift in f.1, towards more positive potentials and altered its slope, while producing a small increase in gr but no effect on d2.. In neonatal single cells, ryanodine (10 /tM) had no significant effect on contraction, ICa. do, f., or g9. Caffeine (10 mM), a less specific agent widely used to investigate sarcoplasmic reticulum function, had actions similar to those of ryanodine. 4. In adult myocytes, when EGTA (10 or 20 mM) or bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 10 mm) were included in the pipette solution, contractions were rapidly abolished, while a small (4 mV) shift offo" to more positive potentials was seen. A large additional shift of f. was observed when ryanodine (10 /SM) was added to the superfusion solution in the continued presence of EGTA or BAPTA. The alterations of ICa in EGTA (or BAPTA) plus ryanodine were the same as those seen in ryanodine alone. In neonatal cells, in contrast, when EGTA or BAPTA were included in the pipette solution we observed only a small effect on foc and the application of ryanodine had no effect.5. Electron micrographs of our preparations show that the dissociated adult cells have sharp sarcolemmal borders, fully developed sarcomeres with T-tubules and sarcoplasmic reticulum membranes. In contrast, the neonatal cells that we use have few of these intracellular structures. Our observations in these preparations are consistent with the work of others (e.g. Penefsky, 1974;Hirakow & Gotoh, 1975;Ishikawa & Yamada, 1975;Legato, 1975;Hoerter, Mazet & Vassort, 1981).6. Our data suggest that fully developed sarcoplasmic reticulum in rat heart cells can affect ICa. We discuss these results in terms of two possible explanations: (1) We conclude that some of the differences in ICa measured in rat heart cells during development can be attributed to the development of the sarcoplasmic reticulum and its connections to the T-tubules and sarcolemma via the spanning proteins.

The effect of angiotensin II on cultured neonatal rat heart myocytes was studied by measuring changes in cell length, the magnitude and kinetics of the calcium current, and changes in cyclic adenosine 3',5'-monophosphate (cAMP) and phosphoinositide metabolism. Spontaneous beating frequency of multicellular networks was increased by angiotensin II with a maximal increase of 100% above control values at concentrations of 5 nM or greater. The half-maximal response occurred at 0.6 nM angiotensin II. Shortening amplitude, shortening velocity, and relaxation velocity decreased concomitantly with the increasing contractile rate. In voltage-clamped single myocytes, both steady-state and transient components of the calcium current were increased by the addition of angiotensin II. Angiotensin II had no effect on either control or isoproterenol-stimulated adenylate cyclase activity in myocyte membranes. Neither the basal levels nor the isoproterenol-stimulated cAMP accumulation in intact cells was affected by addition of hormone. In myocytes labeled with [3H]inositol, angiotensin II stimulated the formation of [3H]inositol phosphates. One minute after addition of 5 nM angiotensin II, inositol monophosphate and inositol bisphosphate levels were increased to 73% and 99%, respectively, above control values and remained elevated at 10 minutes. Inositol trisphosphate levels were not significantly different from control values at either time point. Nifedipine (10 microM) had no effect on angiotensin II-induced increases in [3H]inositol phosphates. We conclude that the increases in both spontaneous beating rate and calcium current in angiotensin II-stimulated cultured neonatal heart cells are not dependent on cAMP or inositol trisphosphate levels but may involve sustained phosphoinositide hydrolysis.

under reduced pressure. Preparative thin layer chromatography of the residue (silica gel eluted with 4:1 benzene-ethyl acetate) resulted in the isolation of 25 mg of the major component. When quickly recrystallized from methanol, fine crystalline material was obtained, mp 180-193°slow decomposition, then rapid gas evolution: uv max 229 nm (e 1.1 X 104) and shoulder 260 nm (e 5 X 103);

The effects of increased protein kinase C activity were studied in neonatal rat myocytes grown in primary culture. The changes in mechanical and electrical behavior, as well as protein phosphorylation, that followed the apparent activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined. As spontaneous beating frequency was increased minimally by 10 nM TPA and by 100% with 85 nM TPA, shortening amplitude, shortening velocity, and relaxation velocity decreased concomitantly. In contrast, 4-alpha-phorbol-12,13-didecanoate (alpha-PDD), which does not activate protein kinase C, had no effect on beating behavior at 800 nM. In voltage-clamped single myocytes, both steady-state and transient components of the cadmium-sensitive calcium current were increased by the addition of TPA (65 nM). Neither the time constant for the inactivation of the transient component of this calcium current nor the reversal potential was altered by TPA. The phosphorylation state of a discrete set of proteins, with apparent molecular weights of 32 and 83 kDa, was enhanced when TPA was added to intact myocytes. Angiotensin II enhances the phosphorylation state of the same set of proteins as observed with TPA. We conclude that activation of protein kinase C can modify mechanical behavior and increase L-type Ca2+ channel activity in cultured neonatal rat ventricular myocytes. The remarkable similarity in mechanical, electrical, and protein phosphorylation responses of cultured neonatal myocytes following TPA or angiotensin II application indicate that protein kinase C may mediate the action of angiotensin II.

1. Calcium currents (ICa) from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the 'whole‐cell' voltage‐clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Examination of ICa was limited to one calcium channel type, 'L' type (Nilius, Hess, Lansman & Tsien, 1985), by appropriate voltage protocols. 2. We measured transient and steady‐state components of ICa, and could generally describe ICa in terms of the steady‐state activation (d infinity) and inactivation (f infinity) parameters. 3. We observed that the reduction of ICa by the calcium channel antagonist D600 can be explained by both a shift of d infinity to more positive potentials as well as a slight reduction of ICa conductance. D600 did not significantly alter either the rate of inactivation of ICa or the voltage dependence of f infinity. 4. The calcium channel modulator BAY K8644 shifted both d infinity and f infinity to more negative potentials. Additionally, BAY K8644 increased the rate of inactivation at potentials between +5 and +55 mV. Furthermore, BAY K8644 also increased ICa conductance, a change consistent with a promotion of 'mode 2' calcium channel activity (Hess, Lansman & Tsien, 1984). 5. We conclude that, as predicted by d infinity and f infinity, there is a significant steady‐state component of ICa ('window current') at plateau potentials in neonatal rat heart cells. Modulation of the steady‐state and transient components of ICa by various agents can be attributed both to specific alterations in d infinity and f infinity and to more complicated alterations in the mode of calcium channel activity.