The function of the heart depends critically on an adequate oxygen supply through the coronary arteries. Coronary arteries dilate when the intravascular oxygen tension decreases. Hypoxic vasodilation in isolated, perfused guinea pig hearts can be prevented by glibenclamide, a blocker of adenosine triphosphate (ATP)-sensitive potassium channels, and can be mimicked by cromakalim, which opens ATP-sensitive potassium channels. Opening of potassium channels in coronary smooth muscle cells and the subsequent drop in intracellular calcium is probably the major cause of hypoxic and ischemic vasodilation in the mammalian heart.

SUMMARY1. Primary cultures of coronary endothelial cells were obtained by enzymatic dispersion of isolated guinea-pig hearts and separation of different cardiac cell types by density gradient centrifugation. The cells were grown to confluency and the membrane potential of the monolayer was recorded using the whole-cell-clamp mode of the patch-clamp technique.2. When superfused with physiological salt solution at 37°C the average resting potential of the monolayers was -35 + 9 mV. A 2 min application of bradykinin (0-1-20 nm) induced a transient hyperpolarization of up to 40 mV (median 33 mV), which was followed by a sustained depolarization of up to 28 mV (median 10 mV). The average duration of the hyperpolarization, measured midway between the resting potential and peak negativity, was 48 s with 20 nM-bradykinin.3. The concentration of bradykinin producing a half-maximal hyperpolarization was 2-5 nm. When high concentrations of bradykinin (> 10 nM) were applied for several minutes voltage oscillations of low amplitude with periodicity of 2-3 min were observed.4. The peak of the hyperpolarization depended on the extracellular potassium concentration ([K+] [K+]. the hyperpolarization turned into a depolarization.5. After removal of external Ca2+ the first transient hyperpolarization elicited by bradykinin had the same amplitude as under control conditions, but its duration was reduced to about 72%. The second application of bradykinin in Ca2+-free solution produced only a depolarization. The hyperpolarizing response to bradykinin could be re-primed by exposing the preparation to Ca2+-containing solution for 2 min.6. The transient hyperpolarization elicited by 4 nM-bradykinin could be inhibited by d-tubocurarine, a blocker of Ca2+-activated potassium channels. On average, 1 mM-tubocurarine reduced the hyperpolarization by 49+18%. Apamin (10 /M) reduced the hyperpolarization by 15 + 11 %.7. ATP (1 ,/M) produced a hyperpolarization of similar amplitude to that produced by bradykinin, but of shorter duration (average 29 s), and a very small (< 5 mV) or MS 8342 G. MEHRKE AND J. DA UT no sustained depolarization. Histamine (10 /LM) produced an even shorter transient hyperpolarization, followed by a depolarization of up to 15 mV.8. Most of the monolayers of coronary endothelial cells responded to adenosine in a similar way as to bradykinin. The hyperpolarization induced by adenosine (1 /M) was abolished in the presence of 50 ,uM-theophylline, an antagonist of P1 purinoceptors. The response to ATP was unaffected by theophylline.9. Phorbol dibutyrate (2 gUM) produced a sustained depolarization of about 5 mV. During the sustained depolarization produced by a maximally effective concentration of bradykinin, addition of ATP or adenosine could still produce nearly the same hyperpolarizing response as under control conditions. 10. Our results suggest that endothelium-dependent vasodilators induce a secondmessenger-operated release of Ca2+ from intracellular stores, followed by an increase in Ca21 influx from the extracellu...

The Ca2+ channel subunits alpha 1C-a and alpha 1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba2+ current (IBa) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 microM cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 microM PKA and 1 microM okadaic acid. The activity of the alpha 1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 microM H 89 and was not increased by superfusion with 5 microM forskolin plus 20 microM isobutyl-methylxanthine (IBMX). The alpha 1C-a.beta 2.alpha 2/delta complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 microM H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the alpha 1C-a.beta 2.alpha 2/delta channel with 10 microM PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca2+ current (ICa) of cardiac myocytes increased threefold during internal dialysis with 5 microM PKA or 25 microM microcystin and during external superfusion with 0.1 microM isoproterenol or 5 microM forskolin plus 50 microM IBMX. These results indicate that the L-type Ca2+ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.

SUMMARY1. Coronary endothelial cells were isolated from adult guinea-pig hearts (Nees, Gerbes & Gerlach, 1981) and the electrical properties of primary cultures were studied using the tight-seal whole-cell recording mode of the patch clamp technique.2. On the third or fourth day in culture whole-cell clamp records from single coronary endothelial cells were obtained at 37 'C. The resting potential was -33 + 6 mV (n = 10). The membrane time constant determined with rectangular current pulses was 68 + 22 ms (n = 10).3. In voltage clamp experiments, no time-dependent membrane conductance changes were found in the range -80 to + 40 mV. The current-voltage relation was linear in normal physiological salt solution containing 5-4 mM-K+. The input resistance was 1P7 +0 4 GQ. When the external K+ concentration was increased to 116 mm the cells depolarized to about -3 mV and the clamp currents showed marked inward rectification.4. Between days four and seven in culture the endothelial cells formed confluent monolayers which showed the characteristic 'cobblestone' morphology. The input resistance of cells in a monolayer was 8 + 3 MQ, i.e. a factor of 200 lower than that found in single cells. It was concluded that the cells in the confluent monolayer are coupled electrically by gap junctions.5. Exposure of coronary endothelial cells to K+-free solution for 5 min produced a depolarization of about 8 mV. Upon readmission of normal external K+ the cells transiently hyperpolarized by up to 20 mV. This transient hyperpolarization decayed with a time constant of 1-9 + 0-3 min.6. The transient hyperpolarization could be abolished by application of 2 x 10-4 Mdihydro-ouabain (DHO). Application of DHO in the steady state produced a depolarization of 8+1 mV. From these findings it was concluded that coronary endothelial cells possess an electrogenic sodium pump which contributes about -8 mV to the resting potential.7. From the passive electrical properties of single cells and the morphological data t Present address:

SUMMARY1. The effects of bradykinin, ATP, adenosine, histamine and thrombin on the membrane potential of confluent monolayers of cultured bovine aortic endothelial cells (BAECs) and guinea-pig coronary endothelial cells (GCECs) were studied at 37°C using the whole-cell mode of the patch-clamp technique.2. The amplitude histogram of the resting potentials of BAEC monolayers showed a bimodal distribution with one peak around -25 mV and another peak around -85 mV. Transitions from one potential level to the other were observed. The bistable membrane potential can be explained by an N-shaped current-voltage relation of the endothelial cell membrane.3. When BAECs with a low resting potential (-10 to -30 mV) were superfused with maximally effective concentrations of ATP (2-10 ,tM) an initial hyperpolarization of -80 to -90 mV was observed which decayed to a plateau of about -60 mV within min. When ATP was removed after 2-3 min the membrane potential returned to control level within 1 min. This was followed by a second hyperpolarization of 10-20 mV, which decayed within 15 min.4. In the absence of extracellular calcium, ATP produced only a brief transient hyperpolarization in aortic endothelium. The plateau and the secondary hyperpolarization were abolished. These findings are consistent with the idea that the changes in membrane potential reflect changes in intracellular free Ca2+ and that the initial peak is due to release of Ca2+ from intracellular stores, whereas the plateau and the secondary hyperpolarization depend on transmembrane Ca2+ influx.5. Bradykinin evoked potential changes similar to ATP in BAECs, except that the secondary hyperpolarization during wash-out was absent. When the membrane potential was more negative than -80 mV, ATP and bradykinin induced only a small initial hyperpolarization followed by a depolarization of up to 20 mV.6. In aortic endothelium, ADP (10 /LM) evoked a much smaller response than ATP. G. MEHRKE, U. POHL AND J. DA UT Adenosine (10 fM), thrombin (2 units/ml), acetylcholine (10 JM) and histamine (10 gM) had only a very small effect on the membrane potential, if any.7. The amplitude histogram of the membrane potential of GCECs showed only one peak around -35 mV. In coronary endothelium, application of bradykinin, ATP, histamine, thrombin, acetylcholine and adenosine all evoked a transient hyperpolarization of 10-40 mV lasting 1 min or less, which then turned into a depolarization.8. The K+ channel openers cromakalim (BRL 34915) and lemakalim (BRL 38227) did not affect the membrane potential of GCECs or BAECs. This suggests that ATPsensitive K+ channels are absent in endothelial cells.9. Reducing the oxygen tension (Po2) of the superfusate from 150 mmHg to 12-32 mmHg also had virtually no effect on the resting potential of BAECs and GCECs. However, in sixteen out of twenty-six experiments 15 min hypoxia produced an increase in input resistance; the median of the increase was 9 %. This may be due to partial uncoupling of the cells in the monolayer caused by closure of intercellular ...