Kathleen D. Keef

Cyclic GMP-dependent Protein Kinase Activates Cloned BKCa Channels Expressed in Mammalian Cells by Direct Phosphorylation at Serine 1072

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the α-subunit of the large conductance Ca2+-activated K+ channel,cslo-α, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-α exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) ofcslo-α channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Iα to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-α in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-α by direct phosphorylation at serine 1072.

Regulation of Ca2+ channels by cAMP and cGMP in vascular smooth muscle cells.

Whole-cell Ca2+ channel currents in rabbit portal vein cells were recorded using the amphotericin B-perforated patch-clamp technique at 35 degrees C. This technique allowed recording of stable inward currents in the absence of run-down for more than 30 minutes. Depolarizing voltage steps from a holding potential of -70 mV elicited voltage-dependent inward currents. The voltage dependence of inward currents measured in either 2.5 mmol/L Ba(2+)- or 2.5 mmol/L Ca(2+)-containing solution were very similar. However, maximum Ba2+ current (obtained at around +10 mV) was approximately 1.5-fold larger than maximum Ca2+ current. Changing the holding potential from -70 to -40 mV decreased inward currents but did not shift the voltage dependence significantly. Inward currents were also completely blocked by the dihydropyridine Ca2+ channel blocker, nicardipine (10 mumol/L), suggesting the presence of predominantly L-type Ca2+ channels in rabbit portal vein cells. Isoproterenol caused small increases in the amplitude of Ba2+ currents in a concentration-dependent manner (10 nmol/L to 1 mumol/L), which were reversed with propranolol. Forskolin (1 mumol/L) or 8-bromo-cAMP (0.1 mmol/L) also caused small increases in the amplitude of Ba2+ currents, suggesting that the stimulatory actions of isoproterenol are importantly linked to the production of cAMP. Higher concentrations of of isoproterenol (10 mumol/L) or forskolin (10 mumol/L) caused a transient increase in Ba2+ currents followed by f decrease in current amplitude. Higher doses of 8-bromo-cAMP (1 mmol/L) and low doses of 8-bromo-cGMP (0.1 mmol/L) inhibited Ba2+ currents, increased the rate of current inactivation, and produced a negative voltage shift in steady-state availability. These results indicate that low concentrations of intracellular cAMP produce modest increases in Ca2+ channel activity, whereas cGMP and higher concentrations of cAMP result in inhibition of Ca2+ channel activity in vascular smooth muscle cells. The observed similarities of cGMP and high concentrations of cAMP on Ba2+ current amplitude, kinetics, and steady-state inactivation suggest mediation by a common mechanism, possibly involving activation of cGMP-dependent protein kinase.

Control of motility patterns in the human colonic circular muscle layer by pacemaker activity

1 This study characterized the electrical and mechanical activities of human colonic muscle strips obtained from either the ascending, descending or sigmoid colon of patient volunteers during elective colon resections. 2 Rhythmic contractile activity was observed in colonic circular muscle strips in the absence of external stimuli. This activity persisted in the presence of atropine, phentolamine, propranolol, tetrodotoxin and Nω‐nitro‐L‐arginine but was abolished by nifedipine. 3 The activity of whole circular muscle (WCM) was compared with that of the myenteric half (MCM), the submucosal half (SCM) and the interior (ICM) of the circular muscle layer. WCM exhibited a prominent 2–4 contractions min−1 contractile pattern which was also present in strips of SCM. In contrast, MCM and ICM exhibited slow (0.3–0.6 contractions min−1), long duration contractions with superimposed higher frequency contractions (17–18 contractions min−1). 4 Resting membrane potential (Vm), recorded at various positions through the thickness of WCM strips did not differ and averaged −50 mV. 5 Slow waves were observed in 83 % of muscles. They averaged 12 mV in amplitude, 9.4 s in duration and had a frequency of 2–4 contractions min−1. Slow waves were greatest in amplitude near the submucosal edge and decreased with distance away from this edge. Each slow wave was associated with a transient contraction. 6 Near the myenteric edge, rapid fluctuations of Vm with a mean frequency of 18 contractions min−1 were recorded in 67 % of muscles. Spiking activity was common and was superimposed upon slow waves and rapid Vm fluctuations. 7 In summary, slow waves were identified in the human colonic circular muscle layer which arise at or near the submucosal edge. These electrical events give rise to a 2–4 contractions min−1 contractile rhythm which is characteristic of the intact muscle layer. Thus, the nature and spatial organization of pacemaker activity in the human colon bears significant resemblance to other animal models, such as the dog and pig.

Involvement of cyclic GMP in non‐adrenergic, non‐cholinergic inhibitory neurotransmission in dog proximal colon

1 Nitric oxide (NO) may serve as a non‐adrenergic, non‐cholinergic (NANC) neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. We tested whether guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) may serve as a second messenger in transducing the NO signal into inhibitory junction potentials (i.j.ps) and relaxation in the canine proximal colon. 2 The membrane permeable analogue of cyclic GMP, 8‐bromo cyclic GMP (8‐Br‐cyclic GMP) mimicked the effects of NO by hyperpolarizing cells near the myenteric border of the circular muscle layer and shortening slow waves in cells near the submucosal surface of the circular muscle layer. 8‐Br‐cGMP also inhibited spontaneous phasic contractions. 3 The specific cyclic GMP phosphodiesterase inhibitor, M&B 22948, hyperpolarized cells near the myenteric border and prolonged the duration of i.j.ps. M&B 22948 also inhibited phasic contractile activity. 4 Methylene blue failed to reduce significantly the amplitude and duration of i.j.ps and had variable effects on contractions. 5 Cyclic GMP levels were assayed in unstimulated muscles and in muscles exposed to exogenous NO and electrical field stimulation. Both stimuli hyperpolarized membrane potential, inhibited contractions, and elevated cyclic GMP levels. 6 Treatment of muscles with l‐NG‐nitroarginine methyl ester (l‐NAME) increased spontaneous contractile activity and lowered cyclic GMP levels. The inhibitory effect of M&B 22948 on contractions was greatly reduced after muscles were treated with l‐NAME. 7 These data support the concept that the effects of NANC nerve stimulation and NO (which may be one of the enteric inhibitory transmitters) may be mediated by cyclic GMP.

Endothelium-dependent relaxation and hyperpolarization in guinea-pig coronary artery: role of epoxyeicosatrienoic acid

Acetylcholine (ACh) elicits an endothelium‐dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea‐pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial‐derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 μM Nω‐nitro‐L‐arginine and 10 μM indomethacin. ACh, AA and 11,12‐EET each produced concentration‐dependent relaxations in arteries contracted with the H1‐receptor agonist AEP (2,2‐aminoethylpyridine). The AA‐induced relaxation was significantly enhanced in the presence of the cyclo‐oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 μM). The cytochrome P450 inhibitors proadifen (10 μM) and clotrimazole (10 μM) inhibited ACh, lemakalim (LEM) and AA‐induced relaxation, whereas 17‐octadecynoic acid (100 μM) and 7‐ethoxyresorufin (10 μM) were without effect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 μM) and LEM (1 μM)‐induced hyperpolarization. The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12‐EET was also determined. Iberiotoxin (IBTX; 100 nM) was without effect on responses to ACh but significantly reduced responses to both AA and 11,12‐EET. In contrast, 4‐aminopyridine (4‐AP; 5 mM) significantly reduced response to ACh but not responses to AA and 11,12‐EET. Combined IBTX plus (4‐AP) inhibited the ACh‐induced relaxation to a greater extent than 4‐AP alone. Apamin (1 μM), glibenclamide (10 μM) and BaCl2 (50 μM) had no significant effect on responses to ACh, AA and 11,12‐EET. IBTX (100 nM) significantly reduced both 11,12‐EET (33 μM) and AA (30 μM) hyperpolarization without affecting the ACh (1 μM)‐induced hyperpolarization. In contrast, 4‐AP significantly reduced the ACh‐induced hyperpolarization without affecting either AA or 11,12‐EET‐induced hyperpolarizations. In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12‐EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12‐EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO‐ and prostaglandin‐independent hyperpolarization and relaxation in the GPCA.

Modulation of K+ and Ca2+ channels by histamine H1-receptor stimulation in rabbit coronary artery cells.

1. The modulation of whole‐cell K+ and Ca2+ currents by stimulation of histamine H1‐receptors in freshly isolated single smooth muscle cells from the rabbit coronary artery was characterized using the patch‐clamp technique at 35 degrees C. Single‐channel K+ currents were also analysed using the cell‐attached patch configuration. 2. The histamine H1‐receptor agonist, 2‐(2‐aminoethyl)pyridine (AEP) (0.1 mM), increased the amplitude of voltage‐activated inward Ba2+ currents, recorded using the perforated‐patch recording technique, which could be completely blocked by the dihydropyridine antagonist, nicardipine (1 microM). 3. Whole‐cell outward K+ currents in rabbit coronary artery cells could be classified into at least two components: (a) a slowly inactivating, 4‐aminopyridine (4‐AP)‐sensitive low‐noise current, and (b) a non‐inactivating, tetraethylammonium (TEA)‐sensitive high‐noise current. 4. AEP (0.1 mM) caused changes in whole‐cell outward K+ currents which depended upon membrane voltage. Specifically: (a) AEP enhanced the amplitude of outward currents at voltages between ‐30 and 0 mV, and (b) AEP decreased the outward currents at more positive potentials. 5. The removal of extracellular Ca2+ caused little inhibition of the effects of AEP on K+ currents, whereas the depletion of intracellular Ca2+ stores by pretreatment with ryanodine and caffeine prevented the effects of AEP on K+ channels. Moreover, acute exposure to ryanodine (10 microM) or thapsigargin (1 microM), a Ca(2+)‐ATPase inhibitor, caused voltage‐dependent changes in the outward currents similar to those observed with AEP. These results suggest that the voltage‐dependent effects of AEP on K+ currents are mainly mediated by release of Ca2+ from intracellular stores. 6. The dual stimulatory and inhibitory effect of AEP on whole‐cell K+ currents was shown to be due to a differential effect on two distinct types of K+ channels. The stimulatory effect observed over the voltage range ‐30 to 0 mV was prevented by pretreatment of cells with low concentrations of TEA (1 mM), whereas the inhibitory effect observed at positive potentials was prevented by pretreatment of cells with 4‐AP (3 mM). 7. Single‐channel recordings revealed two types of unitary K+ currents with conductances of 225 and 70 pS in the cell‐attached configuration with symmetrical K+ solutions (150 mM K+ in pipette‐150 mM K+ in bath). Bath application of AEP (0.1 mM) caused a marked increase in the open probability of the large conductance channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular divalent cations block smooth muscle K+ channels.

The patch-clamp technique was used to examine the sensitivity of delayed rectifier K+ channels to changes in intracellular divalent cations (Mg2+ and Ca2+). During voltage-step and ramp depolarizations, a delayed rectifier K+ current (IK(dr)) was identified in renal, pulmonary, coronary, and colonic smooth muscle cells as a low-noise outward current that activated near -40 mV, was sensitive to 4-aminopyridine (4-AP), and was insensitive to charybdotoxin. During whole-cell voltage-clamp experiments in each of the cell types, the 4-AP-sensitive IK(dr) was significantly less in cells dialyzed with 10 mM Mg2+ as compared with cells in which no Mg2+ was added to the internal dialysis solution (P < or = .05, n > or = 4). In coronary artery cells, 100 microM 2-(2-aminoethyl)pyridine (an H1 receptor agonist) or 10 microM ryanodine, agents that cause an increase in [Ca2+]i, also caused a significant reduction of the 4-AP-sensitive IK(dr) similar to that produced by Mg2+. 4-AP (5 mM) significantly depolarized single renal arterial cells that were dialyzed with Mg(2+)-free solution but not those dialyzed with 10 mM Mg2+ (P < .01, n = 4). In inside-out patches of renal arterial smooth muscle cells, with 200 nM charybdotoxin in the patch pipette to block large conductance Ca(2+)-activated K+ channels, a 59 +/- 10-picosiemen K+ channel that was sensitive to cytoplasmic Mg2+ was identified. In Mg(2+)-free solution, channel open probability was 0.028 +/- 0.012 (n = 8) and 0.095 +/- 0.011 (n = 8) at +40 and +80 mV, respectively. When the bath solution was changed to one containing 5 or 15 mM Mg2+, channel open probability was significantly reduced by 66% and 68% (+40 mV) or 93% and 96% (+80 mV), respectively. This decrease in the open probability of the delayed rectifier K+ channel resulted from a concentration- and voltage-dependent decrease in mean open time. At +40 mV, time constants for the open time distribution were significantly decreased from 5.5 +/- 0.52 to 1.2 +/- 0.14 milliseconds, whereas the closed time constant was significantly increased from 634 +/- 11.1 to 820 +/- 14.4 milliseconds (P < .01, n = 4). It is concluded that a 4-AP-sensitive delayed rectifier K+ channel in both vascular and visceral smooth muscle cells is modulated by changes in intracellular Ca2+ and Mg2+ that may alter membrane potential and the contractile state of smooth muscle.

Basal release of nitric oxide induces an oscillatory motor pattern in canine colon.

1. The consequences of intrinsic, basal nitric oxide release on electrical and contractile activity of canine proximal colon were examined. Membrane potential and contraction were simultaneously recorded from the circular muscle in the presence of drugs to block adrenergic and cholinergic responses. 2. Electrical slow waves were recorded from muscle cells near the submucosal surface of the circular layer. Spontaneous contractions were initiated by each slow wave. Contractile amplitude increased 1.9‐fold when nerves were blocked with tetrodotoxin (TTX, 1 microM). 3. Muscle cells near the myenteric surface displayed myenteric potential oscillations (MPOs) averaging 16 cycles per minute (c.p.m.) in frequency and 10 mV in amplitude. Twenty‐five per cent of muscles displayed an additional slow, neurogenic oscillation (mean frequency, 1 c.p.m.; amplitude, 14 mV) superimposed upon the MPO rhythm. 4. The nitric oxide (NO) synthase inhibitor N omega ‐nitro‐L‐arginine (L‐NA, 100 microM; n = 16) abolished neurogenic oscillations, depolarized cells, and increased MPO upstroke velocity, amplitude and frequency. The actions of L‐NA were mimicked by N omega‐nitro‐L‐arginine methylester (L‐NAME, 100 microM) and oxyhaemoglobin (3%). 5. Spontaneous contractions were increased 2.3‐fold by L‐NA, and TTX had no effect on contractions after addition of L‐NA. 6. The NO‐donor sodium nitroprusside (SNP, 1 microM) reversed the electrical and mechanical effects of L‐NA and initiated slow oscillations similar to the neurogenic oscillations. Slow oscillations were also evoked with S‐nitroso‐N‐acetylpenicillamine (SNAP, 1 microM). The effects of NO donors were blocked by oxyhaemoglobin. 7. Slow electrical oscillations could not be elicited by SNP after removal of a thin strip of circular muscle along the myenteric edge. 8. These data suggest that the spontaneous electrical and contractile activity of the proximal colon is tonically suppressed by basal release of NO. Basal NO causes an oscillatory pattern of electrical and mechanical activity. This activity does not require patterned firing of nerves; rather a continuous, low level release of NO would be capable of producing the neurogenic oscillatory behaviour. The slow oscillatory activity depends upon the presence of the myenteric region of the circular muscle layer, which contains cell bodies of enteric neurons and interstitial cells of Cajal.

Comparison of the actions of acetylcholine and BRL 38227 in the guinea-pig coronary artery.

1 The contractile and electrical responses to acetylcholine (ACh) in isolated segments of guinea‐pig and rabbit coronary arteries were compared to those of the putative adenosine 5′‐triphosphate (ATP)‐dependent K+ channel opener, BRL 38227. 2 Both ACh and BRL 38227 produced concentration‐dependent relaxation of vessel segments contracted with the H1‐receptor agonist, 2‐(2‐aminoethyl)pyridine. 3 An IC90 of either vasodilator also produced 17–20 mV of hyperpolarization of the guinea‐pig coronary artery. 4 Glibenclamide (1–35 μm) depolarized the guinea‐pig coronary artery by 8–12 mV and antagonized BRL 38227‐ but not ACh‐induced relaxation and hyperpolarization. 5 In the guinea‐pig coronary artery, the K+ channel blockers phencyclidine (PCP, 100 μm), tetraethylammonium (TEA, 10 mm) and scorpion venom (8.7 μg ml−1) all significantly reduced ACh‐induced relaxation and hyperpolarization whereas only PCP was an effective antagonist of both relaxation and hyperpolarization with BRL 38227. 6 Similar effects of glibenclamide and scorpion venom on ACh‐ and BRL 38227‐induced relaxation were observed in the rabbit coronary artery. 7 Apamin (3.5 μm) was without effect on either the ACh‐ or BRL 38227‐induced relaxation in the guinea‐pig coronary artery. 8 In conclusion, the actions of BRL 38227 in coronary artery are compatible with its proposed effects on ATP‐dependent K+ channels. In contrast, the results with ACh suggest that some step between the initial binding of ACh to endothelial muscarinic receptors and the final relaxation of the smooth muscle depends upon the opening of Ca2+‐activated K+ channels.

Muscarinic M2 Receptor Stimulation of Cav1.2b Requires Phosphatidylinositol 3-Kinase, Protein Kinase C, and c-Src

Abstract— This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 &mgr;mol/L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4-DAMP (100 nmol/L) but was blocked by the M2 receptor antagonist methoctramine (5 &mgr;mol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 &mgr;mol/L), by the c-Src inhibitor PP2 (10 &mgr;mol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit G&bgr;&ggr;. Cells dialyzed with c-Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c-Src is downstream of PI3K. Phorbol 12,13-dibutyrate (PDBu, 0.1 &mgr;mol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to G&bgr;&ggr;, PI3K, a novel PKC, and c-Src. PDBu stimulates the novel PKC/c-Src pathway along with a second pathway that is independent of c-Src and involves a classical PKC.