Coupling an HCN2-expressing cell to a myocyte creates a two-cell pacing unit.

We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell-cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell-cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of approximately 0.6 to 1.7 Hz (1.09 +/- 0.05 Hz). Addition of carbenoxolone (200 microM), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 microM 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.

Pacemaker current and automatic rhythms: toward a molecular understanding.

The ionic basis of automaticity in the sinoatrial node and His-Purkinje system, the primary and secondary cardiac pacemaking regions, is discussed. Consideration is given to potential targets for pharmacologic or genetic therapies of rhythm disorders. An ideal target would be an ion channel that functions only during diastole, so that action potential repolarization is not affected, and one that exhibits regional differences in expression and/or function so that the primary and secondary pacemakers can be selectively targeted. The so-called pacemaker current, If, generated by the HCN gene family, best fits these criteria. The biophysical and molecular characteristics of this current are reviewed, and progress to date in developing selective pharmacologic agents targeting If and in using gene and cell-based therapies to modulate the current are reviewed.

Molecular/genetic determinants of repolarization and their modification by environmental stress.

Although a variety of factors, inherited or environmental, can influence expression of ion channel proteins to impact on repolarization, that environment can affect genetic determinants of repolarization for intervals of varying duration is a concept that is not as generally appreciated as it should be. In the following pages we review the molecular/genetic determinants of cardiac repolarization and summarize how pathologic events and environmental intrusions can affect these determinants. Understanding the chains of events involved should yield insights into both the causes and potential avenues of treatment for abnormalities of repolarization.

Transmural gradients in Na/K pump activity and [Na+]I in canine ventricle.

There are well-documented differences in ion channel activity and action potential shape between epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) ventricular myocytes. The purpose of this study was to determine if differences exist in Na/K pump activity. The whole cell patch-clamp was used to measure Na/K pump current (I(P)) and inward background Na(+)-current (I(inb)) in cells isolated from canine left ventricle. All currents were normalized to membrane capacitance. I(P) was measured as the current blocked by a saturating concentration of dihydro-ouabain. [Na(+)](i) was measured using SBFI-AM. I(P)(ENDO) (0.34 +/- 0.04 pA/pF, n = 17) was smaller than I(P)(EPI) (0.68 +/- 0.09 pA/pF, n = 38); the ratio was 0.50 with I(P)(MID) being intermediate (0.53 +/- 0.13 pA/pF, n = 19). The dependence of I(P) on [Na(+)](i) or voltage was essentially identical in EPI and ENDO (half-maximal activation at 9-10 mM [Na(+)](i) or approximately -90 mV). Increasing [K(+)](o) from 5.4 to 15 mM caused both I(P)(ENDO) and I(P)(EPI) to increase, but the ratio remained approximately 0.5. I(inb) in EPI and ENDO were nearly identical ( approximately 0.6 pA/pF). Physiological [Na(+)](i) was lower in EPI (7 +/- 2 mM, n = 31) than ENDO (12 +/- 3 mM, n = 29), with MID being intermediate (9 +/- 3 mM, n = 22). When cells were paced at 2 Hz, [Na(+)](i) increased but the differences persisted (ENDO 14 +/- 3 mM, n = 10; EPI 9 +/- 2 mM, n = 10; and MID intermediate, 11 +/- 2 mM, n = 9). Based on these results, the larger I(P) in EPI appears to reflect a higher maximum turnover rate, which implies either a larger number of active pumps or a higher turnover rate per pump protein. The transmural gradient in [Na(+)](i) means physiological I(P) is approximately uniform across the ventricular wall, whereas transporters that utilize the transmembrane electrochemical gradient for Na(+), such as Na/Ca exchange, have a larger driving force in EPI than ENDO.

Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions.

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase beta (pol beta) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol beta was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mbeta16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mbeta16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol beta. These two pol beta knockdown cell lines (NRK-kcdc and Mbeta16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mbeta16tsA-wt cells or N2A cells. The levels of pol beta mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mbeta16tsA-kcdc cells with Mbeta16tsA-wt, N2A or NRK-wt cells had no effect on pol beta levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol beta levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol beta in the wt cells. The inability of Mbeta16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.

HCN2 overexpression in newborn and adult ventricular myocytes: distinct effects on gating and excitability.

Ventricular pacemaker current (I(f)) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle. However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular I(f), exhibit only a modest difference in activation voltage. We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. The expressed current exhibited an 18-mV difference in activation (V(1/2) -95.9+/-1.9 in adult; -77.6+/-1.6 mV in neonate), comparable to the 22-mV difference between native I(f) in adult and neonatal cultures (V(1/2) -98.7 versus -77.0 mV). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88+/-5 versus 48+/-4 bpm). In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149+/-266 versus 3140+/-279 mV. ms). Thus, ventricular maturational state influences the voltage dependence of expressed HCN2, resulting in distinct physiological impact of expressed channels in neonate and adult myocytes. The full text of this article is available at http://www.circresaha.org.

MinK-related peptide 1: A beta subunit for the HCN ion channel subunit family enhances expression and speeds activation.

The HCN family of ion channel subunits underlies the currents I(f) in heart and I(h) and I(q) in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a beta subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a beta subunit for the cardiac delayed rectifier I(Kr), these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.

Myocardial blood-flow response during mental stress in patients with coronary artery disease.

Positron emission tomography was used to quantify changes in myocardial blood flow during mental stress in patients with and without coronary artery disease. Blunted augmentation of myocardial blood flow during mental stress was observed in regions without significant epicardial stenosis.

Sympathetic innervation alters activation of pacemaker current (If) in rat ventricle.

Pacemaker current (If) exists in both neonatal and adult ventricles, but activates at more negative voltages in the adult. This study uses whole-cell patch clamp to investigate the factors that may contribute to the maturational shift of If, comparing neonatal rat ventricular myocytes that were cultured for 4-6 days either alone, in co-culture with sympathetic nerves, or with neurotransmitters chronically present in culture. If recorded from nerve-muscle co-cultures had a significantly more negative and shallower activation-voltage relation than that from control muscle cultures, which was reflected in the midpoint potential (V50) and slope factor (K) of activation. This effect of innervation was prevented by the sustained presence in the culture of the alpha1-adrenergic antagonist prazosin (Pz) at 10(-7) M. In parallel experiments, myocytes treated with noradrenaline (NA) at 10(-7) M or neuropeptide Y (NPY) at 10(-7) M during culture had the same If activation as control cells, but cells treated with NA and NPY together had a significantly more negative and shallower activation curve. Maximum conductance and reversal potential were unchanged. The effect of chronic exposure to NA + NPY was prevented by the sustained presence of either Pz or the NPY Y2 selective antagonist T4-[NPY(33-36)]4 (3.5 x 10(-7) M) in the culture, indicating a requirement for both alpha1-adrenergic and NPY Y2 activation. Substituting NA with the alpha1A-adrenergic selective agonist A61603 (5(-10) x 10(-9) M), in the presence of NPY, did not alter If, suggesting the involvement of alpha1B- rather than alpha1A-adrenoceptors. Further, sequential exposure to NPY followed by NA was effective in reproducing the action of chronic simultaneous exposure to these agonists, but sequential exposure to NA followed by NPY was ineffective. The results are consistent with past studies indicating that NPY affects the functional expression of the alpha1B-adrenergic cascade and suggest that sympathetic innervation induces a negative shift of If in ventricle via a combined action at alpha1B-adrenergic and NPY Y2 receptors. This effect of innervation probably contributes to the developmental maturation of If activation.

Effects of the renin-angiotensin system on the current I(to) in epicardial and endocardial ventricular myocytes from the canine heart.

The Ca(2+)-independent portion of transient outward K(+) current (I(to)) exhibits a transmural gradient in ventricle. To investigate control mechanisms for this gradient, we studied canine epicardial and endocardial ventricular myocytes with use of the whole-cell patch-clamp technique. I(to) was larger in amplitude, had a more negative voltage threshold for activation, and had a more negative midpoint of inactivation in epicardium. Recovery from inactivation was >10-fold slower in endocardium. Incubation of epicardial myocytes with angiotensin II for 2 to 52 hours altered I(to) to resemble unincubated endocardium and reduced the amplitude of the phase 1 notch of the action potential. In contrast, incubation of endocardial myocytes with losartan for 2 to 52 hours altered I(to) to resemble unincubated epicardium and induced a phase 1 notch in the action potential. With RNase protection assays, we determined that incubations with angiotensin II or losartan did not alter mRNA levels for either Kv4.3 or Kv1.4; thus, a change in the alpha subunit for I(to) is unlikely to be responsible. To test whether posttranslational modification produced the effects of angiotensin II, we coexpressed Kv4.3 and the angiotensin II type 1a receptor in Xenopus oocytes. Incubation with angiotensin II increased the time constant for recovery from inactivation of the expressed current by 2-fold with an incubation time constant of 3.7 hours. No effect on activation or inactivation voltage dependence was observed. These results demonstrate that the properties of I(to) in endocardium and epicardium are plastic and likely under the tonic-differing influence of the renin-angiotensin system.

Molecular basis for differential sensitivity of KCNQ and I(Ks) channels to the cognitive enhancer XE991.

Channels formed by coassembly of the KCNQ1 (KvLQT1) subunit and the minK subunit underlie slowly activating cardiac delayed rectifier (I(Ks)) in the heart, whereas two other members of the KCNQ channel family, KCNQ2 and KCNQ3, coassemble to underlie the M current in the nervous system. Because of their important physiological function, KCNQ channels have potential as drug targets, and an understanding of possible mechanisms that would enable tissue-specific targeting of these channels will be of significant value to drug development. In this study, we examined the role of the minK subunit in determining the response of KCNQ1 channels to blockade by the cognitive enhancer XE991. Coexpression with minK markedly decreased the sensitivity of KCNQ1 to blockade by XE991. When measured at the end of a 500-ms step, XE991 blockade of the KCNQ1+minK current had a K(D) value of 11.1 +/- 1.8 microM, approximately 14-fold less sensitive than the block of the KCNQ1 current (K(D) = 0.78 +/- 0.05 microM). In addition, XE991 reduced activation and deactivation time constants and caused a rightward shift in the activation curve of KCNQ1+minK, but affected none of these parameters for KCNQ1 alone. Also, XE991 block of KCNQ1+minK, but not of KCNQ1, was time- and voltage-dependent. We conclude that the presence of minK in the I(Ks) channel complex gives rise to differential sensitivity of KCNQ and I(Ks) channels to blockade by XE991. Our results have implications for drug development by demonstrating the important potential role of accessory subunits in determining the pharmacological properties of KCNQ channels.

Felodipine improves left ventricular emptying in patients with chronic heart failure: V-HeFT III echocardiographic substudy of multicenter reproducibility and detecting functional change.

BACKGROUND: The echocardiographic substudy of the Vasodilator-Heart Failure Trial III (V-HeFT III) aimed to determine if felodipine treatment in patients with heart failure who were taking an angiotensin-converting enzyme inhibitor had a favorable effect on left ventricular (LV) structure and function. Earlier V-HeFT trials showed that hydralazine-isosorbide dinitrate improved ejection fraction (EF) and survival, whereas enalapril achieved greater survival with smaller increases in EF. Would the combination of a potent vasodilator and enalapril produce greater improvements in function and survival? METHODS AND RESULTS: Doppler-echocardiographic data were collected from 260 males with heart failure who were randomized to felodipine or a placebo. Mean intrasubject differences between baseline, at 3 months, and at 12 months were compared. Intersite and intrareader reproducibilities were measured from duplicate recordings and readings. At 3 months, no changes in ultrasound variables from baseline occurred in either group. At 12 months, felodipine patients achieved greater increases in EF, shortening of LV end-systolic length, and increases in stroke volume index. Reproducibility coefficients of variation were 7.4% (EF), 6.0% (end-diastolic length), and 13.0% (stroke volume index). CONCLUSIONS: The echocardiographic substudy showed that felodipine, added to heart failure therapy, increased EF, shortened end-systolic length, and increased stroke volume index. The changes were small and confirmed that reproducibility from multiple laboratories can be coordinated into a useful research tool.

Epidermal growth factor increases i(f) in rabbit SA node cells by activating a tyrosine kinase.

Our previous results have demonstrated that tyrosine kinase inhibition reduces i(f) in rabbit SA node myocytes, suggesting that tyrosine kinases regulate i(f). One receptor tyrosine kinase the EGF receptor kinase is known to increase heart rate. To determine if this action is mediated through changes in i(f), we examined the effect of epidermal growth factor (EGF) on i(f) with the permeabilized patch-clamp technique. 0.1 microM EGF increased i(f) amplitude in response to single-step hyperpolarizations in the diastolic range of potentials. This increase was 20+/-3%, n=11 at -75 mV. This effect is caused by activating a tyrosine kinase because 50 microM genistein, a tyrosine kinase inhibitor, eliminated this EGF action. A two-step pulse protocol showed that maximal i(f) conductance was increased by EGF. We further examined this conductance change by constructing the activation curve. The maximal i(f) conductance was increased by 23% with no change in midpoint, V(1/2), control=-74+/-2 mV, V(1/2) EGF=-74+/-1 mV. Thus EGF acts via a tyrosine kinase to increase maximal i(f) conductance with no change in the voltage dependence of activation. These results suggest that EGF effects on i(f) contribute to the positive chronotropic effect of EGF on SA node.

Isoform-Specific Regulation of the Na(+)-K(+) Pump in Heart.

Guinea pig ventricular myocytes coexpress two isoforms of the Na(+)-K(+) pump. These two isoforms respond differently to the physical environment and are coupled to autonomic input through different signal transduction cascades. The expression of different isoforms provides each cell type with a mechanism of programming specific responses to environmental changes.

Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues.

HCN cation channel mRNA expression was determined in the rabbit heart and neonatal and adult rat ventricle using RNase protection assays. In the rabbit SA node, the dominant HCN transcript is HCN4, representing >81% of the total HCN message. HCN1 is also expressed, representing >18% of the total HCN mRNA. Rabbit Purkinje fibers contained almost equal amounts of HCN1 and HCN4 transcripts with low levels of HCN2, whereas rabbit ventricle contained predominantly HCN2. The SA node contained 25 times the total HCN message of Purkinje fibers and 140 times the total HCN message of ventricle. No reports of hyperpolarization-activated current (If) exist in rabbit Purkinje fibers, and we could not record If in rabbit ventricular myocytes. To investigate the possible role of isoform switching in determining the voltage dependence of If, we determined the prevalence of HCN isoforms in neonatal and adult rat ventricle. We had previously determined the threshold for activation of If to be approximately -70 mV in neonatal rat ventricle and -113 mV in adult rat ventricle. In both neonatal and adult rat ventricle, only HCN2 and HCN4 transcripts are present. The ratio of HCN2 to HCN4 is approximately 5:1 in the neonate and 13:1 in the adult. Taken together, these results suggest that different cardiac regions express different isoforms of the HCN family. The HCN1 and HCN4 isoforms are most closely associated with a depolarized threshold for If activation, whereas the HCN2 isoform is associated with a more negative activation curve.

A role for the renin-angiotensin system in the evolution of cardiac memory.

INTRODUCTION: We studied the role of the cardiac renin-angiotensin II system in the genesis of cardiac memory, in which T wave changes induced by ventricular pacing (VP) accumulate and persist during subsequent sinus rhythm. METHODS AND RESULTS: Anesthetized dogs were instrumented via a thoracotomy and three 20-minute runs of VP were interspersed with periods of normal sinus rhythm sufficient to permit T wave recovery to 90% of control. Memory was quantified as the change (delta) in T wave vector angle showing accumulation over the three monitoring periods. In five control dogs T wave vector = -27 +/- 49 degrees, and this shifted by 104 degrees (P < 0.05) over the three postpacing recovery periods. In seven dogs infused with the receptor blocker saralasin, five infused with the angiotensin-converting enzyme inhibitor captopril, and four infused with the tissue protease inhibitor chymostatin, there were significant reductions in the incidence and the accumulation of memory. In four other experiments, we used isolated, blood-perfused canine hearts to demonstrate that VP used to induce memory alters the contractile pattern of the left ventricle. CONCLUSIONS: We propose that the alteration in myocardial stretch induced by pacing activates angiotensin II synthesis by cardiac cells. We propose, further that the endogenous cardiac renin-angiotensin II system (blocked by saralasin, captopril and by chymostatin) is an important contributor to the induction of memory.

Augmented short- and long-term hemodynamic and hormonal effects of an angiotensin receptor blocker added to angiotensin converting enzyme inhibitor therapy in patients with heart failure. Vasodilator Heart Failure Trial (V-HeFT) Study Group.

BACKGROUND: ACE inhibitors may not adequately suppress deleterious levels of angiotensin II in patients with heart failure. An angiotensin receptor blocker added to an ACE inhibitor may exert additional beneficial effects. METHODS AND RESULTS: Eighty-three symptomatic stable patients with chronic heart failure receiving long-term ACE inhibitor therapy were randomly assigned to double-blind treatment with valsartan 80 mg BID, valsartan 160 mg BID, or placebo while receiving their usual ACE inhibitor therapy. Studies were performed before and after the first dose of the test drug and again after 4 weeks of therapy. A single dose of lisinopril was administered during study days to ensure sustained ACE inhibition. Compared with placebo, the first dose of valsartan 160 mg resulted in a significantly greater reduction in pulmonary capillary wedge pressure at 3, 4, and 8 hours and during the prespecified 4- to 8-hour interval after the dose and in systolic blood pressure at 2, 3, 6, 8, and 12 hours and 4 to 8 hours after the dose. A pressure reduction from valsartan 80 mg did not achieve statistical significance. After 4 weeks of therapy, net reductions in 0-hour trough pulmonary capillary wedge pressure (-4.3 mm Hg; P=0. 16), pulmonary artery diastolic pressure (-4.7 mm Hg; P=0.013), and systolic blood pressure (-6.8 mm Hg; P=0.013) were observed in the valsartan 160 mg group compared with placebo. After 4 weeks of therapy, plasma aldosterone was reduced by valsartan 80 mg BID (-52. 1 pg/mL; P=0.001) and 160 mg BID (-47.8 pg/mL; P<0.001) compared with placebo, and there was a trend for a reduction in plasma norepinephrine (-97 pg/mL; P=0.10). Seventy-four of the 83 patients completed the trial. CONCLUSIONS: Physiologically active levels of angiotensin II persist during standard long-term ACE inhibitor therapy.

Transient outward current, Ito1, is altered in cardiac memory.

BACKGROUND: Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing or arrhythmias that persist for variable intervals after resumption of sinus rhythm. METHODS AND RESULTS: We induced long-term cardiac memory (LTM) in conscious dogs by pacing the ventricles at 120 bpm for 3 weeks. ECGs were recorded daily for 1 hour, during which time pacing was discontinued. At terminal study, the heart was removed and the electrophysiology of left ventricular epicardial myocytes was investigated. Control (C) and LTM ECG did not differ, except for T-wave amplitude, which decreased from 0.12+/-0.18 to -0.34+/-0.21 mV (+/-SEM, P<0.05), and T-wave vector, which shifted from -37+/-12 degrees to -143+/-4 degrees (P<0.05). Epicardial action potentials revealed loss of the notch and lengthening of duration at 20 days (both P<0.05). Calcium-insensitive transient outward current (Ito) was investigated by whole-cell patch clamp. No difference in capacitance was seen in C and LTM myocytes. Ito activated on membrane depolarization to -25+/-1 mV in C and -7+/-1 mV (P<0.05) in LTM myocytes, indicating a positive voltage shift of activation. Ito density was reduced in LTM myocytes, and a decreased mRNA level for Kv4.3 was observed. Recovery of Ito from inactivation was significantly prolonged: it was 531+/-80 ms (n=10) in LTM and 27+/-6 ms (n=9) in C (P<0.05) at -65 mV. CONCLUSIONS: Ito changes are associated with and can provide at least a partial explanation for action-potential and T-wave changes occurring with LTM.

Activation of PKC increases Na+-K+ pump current in ventricular myocytes from guinea pig heart.

We have previously shown activation of alpha1-adrenergic receptors increases Na+-K+ pump current (Ip) in guinea pig ventricular myocytes, and the increase is eliminated by blockers of phosphokinase C (PKC). In this study we examined the effect of activators of PKC on Ip. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased IP at each test potential without shifting its voltage dependence. The concentration required for a half-maximal response (K0.5) was 6 microM at 15 nM cytosolic [Ca2+] ([Ca2+]i) and 13 nM at 314 nM [Ca2+]i. The maximal increase at either [Ca2+]i was about 30%. Another activator of PKC, 1, 2-dioctanoyl-sn-glycerol (diC8), increased Ip similarly. The effect of PMA on IP was eliminated by the PKC inhibitor staurosporine, but not by the peptide PKI, an inhibitor of protein kinase A (PKA). PMA and alpha1-adrenergic agonist effects both were sensitive to [Ca2+]i, blocked by PKC inhibitors, unaffected by PKA inhibition, and increased Ip uniformly at all voltages. However, they differed in that alpha1-activation caused a maximum increase of 15% vs 30% via PMA, and alpha1-effects were less sensitive to [Ca2+]i than PMA effects. These results demonstrate that activation of PKC causes an increase in Ip in guinea pig ventricular myocytes. Moreover, they suggest that the coupling of alpha1-adrenergic activation to Ip is entirely through PKC, however alpha1-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist.