3-O-Laurylglyceryl ascorbate reinforces skin barrier function through not only the reduction of oxidative stress but also the activation of ceramide synthesis.

OBJECTIVE: A higher trans-epidermal water loss (TEWL) occurs in rough skin, in elder skin and also in atopic dermatitis. An impaired skin barrier function is considered to be caused by an incomplete construction of the intercellular lamellar structure due to the quantitative reduction of ceramides. Since these symptoms coexist with oxidative stress, we hypothesized that impairment of the skin barrier function is accelerated by oxidative stress. Thus, the purpose of this study was to clarify the effect of oxidative stress on ceramide synthesis and to characterize whether antioxidants can improve skin barrier function. 3-O-Laurylglyceryl ascorbate (VC-3LG), which is a newly amphipathic derivative of ascorbic acid, was evaluated as a candidate antioxidant. METHODS: We characterized the mRNA expression levels of serine palmitoyltransferase (SPT) in normal human epidermal keratinocytes (NHEKs) treated with H2 O2 using real-time PCR analysis. In order to evaluate the effect of VC-3LG on skin barrier function, we used several assays with reconstructed human epidermis equivalents (RHEEs). RESULTS: Ceramide synthesis was down-regulated in NHEKs by oxidative stress. Treatment with VC-3LG abrogated the down-regulation of SPT mRNA in NHEKs caused by oxidative stress, and stimulated SPT mRNA expression levels. In experiments characterizing the antioxidative properties of VC-3LG, VC-3LG reduced oxidative stress in NHEKs by up-regulating catalase mRNA expression. In addition, VC-3LG stimulated the skin barrier function in RHEEs, which had lower TEWL values compared with untreated RHEEs. Furthermore, VC-3LG increased the quantity of ceramide in RHEEs. CONCLUSION: Taken together, we conclude that VC-3LG reinforces the skin barrier function due to its reduction of oxidative stress and its promotion of ceramide synthesis.

Successful treatment of toxoplasmic encephalitis diagnosed early by polymerase chain reaction after allogeneic hematopoietic stem cell transplantation: two case reports and review of the literature.

Toxoplasmic encephalitis represents a rare, but often fatal infection after allogeneic hematopoietic stem cell transplantation. Polymerase chain reaction (PCR)-based preemptive therapy is considered promising for this disease, but is not routinely applied, especially in low seroprevalence countries including Japan. We encountered 2 cases of toxoplasmic encephalitis after transplantation that were successfully treated. The diagnosis of toxoplasmic encephalitis in these cases was confirmed by PCR testing when neurological symptoms were observed. Both patients received pyrimethamine and sulfadiazine treatments within 2 weeks of the development of neurological symptoms, and remained free of recurrence for 32 and 12 months. These results emphasized the importance of the PCR test and immediate treatment after diagnosis for the management of toxoplasmic encephalitis.

Prevalence and clinical significance of supra- or infraclavicular drainage on preoperative lymphoscintigraphy in women with breast cancer.

OBJECTIVE: Preoperative sentinel lymph node (SLN) mapping by lymphoscintigraphy is helpful to evaluate extra-axillary SLNs over a wider range than the blue dye method. However, the clinical value of extra-axillary SLNs remains uncertain. The goal of this study was to determine the prevalence and clinical significance of supra- or infraclavicular drainage on preoperative lymphoscintigraphy in women with breast cancer. MATERIALS AND METHODS: We retrospectively reviewed the files of 942 consecutive breast cancer women who underwent preoperative lymphoscintigraphy for SLN biopsy at our institution between April 2004 and March 2015. RESULTS: Supra- or infraclavicular drainage was detected in 5/942 women (0.5%) on preoperative lymphoscintigraphy. An axillary hot spot was detected in all five women, and a positive axillary SLN was detected in four women. Breast tumor locations were the upper inner or outer quadrants in four women and the lower outer quadrant in one woman. The median follow-up period was 75 months (mean: 92; range: 26-111 months). Recurrence outside the axilla was found in three (60%) women. The woman with a negative SLN status did not undergo adjuvant chemotherapy, but developed extra-axillary lymph node recurrence 3 years after primary surgery. No patient died of metastatic breast cancer at the last follow-up. CONCLUSIONS: The detection of the supra- or infraclavicular SLNs on lymphoscintigraphy may provide additional staging information to tailor individual treatment regimens with regard to the potential risk of recurrence or metastasis of breast cancer.

Cloning of a swelling-induced chloride current related protein from rabbit heart.

Recently, pIcln has been reported to be a regulator of a swelling-induced chloride conductance. We have cloned a cDNA RCL-H1 from rabbit heart, of which primary structure is highly homologous to that of pIcln. Outwardly rectifying currents were recorded in oocytes expressing RCL-H1, which is consistent with the result of pIcln. RNA blot analysis revealed the widespread expression of RCL-H1 mRNA in rabbit tissues. RCL-H1 may play an important role in regulating cell volume and give a clue to revealing molecular structure of swelling-induced chloride channel(s).

Cloning and functional expression of a cardiac inward rectifier K+ channel.

We have isolated a cDNA coding for an inward rectifier K+ channel (RBHIK1) from rabbit heart. The cloned cDNA encodes a protein of 427 amino acids with two putative transmembrane segments. The primary structure of RBHIK1 is highly homologous to that of IRK1 which is an inward rectifier K+ channel recently cloned from mouse macrophage by expression cloning. When expressed in Xenopus oocytes, RBHIK1 current showed strong inward rectification and was inhibited by extracellular Ba2+ and Cs+. RNA blot analysis revealed the expression of RBHIK1 mRNA in various rabbit tissues, especially high level in the ventricular muscle.

The voltage-dependent K+ channel (Kv1.5) cloned from rabbit heart and facilitation of inactivation of the delayed rectifier current by the rat beta subunit.

We have isolated a cDNA coding for a delayed rectifier K+ channel (RBKV1.5) from rabbit heart. The amino acid sequence of RBKV1.5 displays a homology to that of other K+ channels of Kv1.5 class. Overall amino acid identity between RBKV1.5 channel and Kv1.5 channel of other species is about 85%. RNA blot analysis revealed the expression of the primary transcript in various rabbit tissues, at the highest level in both the atrium and ventricle. When expressed in Xenopus oocytes, RBKV1.5 current showed a delayed rectifier type characteristics, which was converted to rapidly inactivating currents upon coexpression with a beta subunit.

Hypertensive episodes and circadian fluctuations of blood pressure in patients with phaeochromocytoma: studies by long-term blood pressure monitoring based on a volume-oscillometric method.

A new portable device for the indirect measurement of arterial blood pressure was successfully applied to detect paroxysmal hypertension and circadian fluctuation of blood pressure in patients with phaeochromocytoma. The device utilizes the volume-oscillometric technique, it is equipped with a microprocessor and allows long-term automatic monitoring of indirect blood pressure in the human finger. Compared with conventional fully automated portable devices of the arm-cuff type, our current equipment is lighter, less noisy, and causes less discomfort. With this device repeated measurements can be made without causing stress or discomfort, and without disturbing sleep. The arterial pressure measurement obtained using this device was reliable and reproducible. In some patients certain symptoms, possibly due to phaeochromocytoma, had been observed for several years before the study, although hypertension had not been identified. While these patients had consistently high circulating catecholamine levels, nocturnal falls in blood pressure were clearly documented. This suggests that plasma catecholamines released from the phaeochromocytoma, though excessive, may be of less physiological importance than other regulatory mechanisms concerned with circadian fluctuation of blood pressure, e.g. the sympathetic nervous system and/or hypothalamo-pituitary-adrenal system. This new device has proved to be a reliable means of monitoring long-term blood pressure and is useful in the diagnosis of paroxysmal hypertension in patients with phaeochromocytoma.

Differences in cardiovascular profile among calcium antagonists.

Calcium antagonists constitute a group of organic compounds with diverse chemical structures. Although their main pharmacodynamic actions are vasodilatation and myocardial depression, they are not uniform in producing these effects. Based on comparison of the potencies in producing negative inotropic, chronotropic and dromotropic effects to the coronary vasodilator potency of individual calcium antagonists determined by use of isolated, blood-perfused papillary muscle, sinoatrial node and atrioventricular node preparations of dogs, calcium antagonists are classified into 2 major groups. The dihydropyridines, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine, PN 200-110 (isradipine) and PY 108-068, are generally far more potent in producing coronary vasodilatation than in producing negative inotropic, chronotropic and dromotropic (first-degree atrioventricular block) effects, although there are minor differences among them. The non-dihydropyridine calcium antagonists, verapamil, diltiazem, KB-944, bepridil and MCI-176, are nearly equipotent in producing coronary vasodilatation and a negative dromotropic effect, although all of them are less potent in producing negative inotropy. The non-dihydropyridine calcium antagonists can be further divided into 2 subgroups. Verapamil, diltiazem and KB-944 are nearly equipotent in producing negative dromotropic and chronotropic effects. Bepridil and MCI-176 are less potent in producing negative chronotropy than in producing negative dromotropy.

Nicorandil as a hybrid between nitrates and potassium channel activators.

Nicorandil, although structurally a nitrate, differs from classic nitrates in several respects. It preferentially dilates resistive vessels. Its effect on venous return in dogs is not unanimously a decrease but rather an increase. In high doses or concentrations it suppresses myocardial contraction and ventricular automaticity, nearly sparing sinoatrial nodal automaticity and atrioventricular nodal conduction. It shortens the effective refractory period of myocardium. These cardiac effects of nicorandil have been explained by its mechanism of action as a potassium (K) channel activator. However, what part of the vascular effects of nicorandil this mechanism is responsible for has not been determined. BRL 34915 and pinacidil, nonnitrate vasodilators with a K-channel activator action, have essentially the same cardiovascular profile as nicorandil in isolated, blood-perfused canine heart preparations. In anesthetized, open-chest dogs the 2 K-channel activators decreased systemic blood pressure and increased venous return and cardiac output without elevating heart rate, unless the cardiodepressant effect emerged. The increase in venous return or cardiac output survived elimination of baroceptor functions. These results taken together with previous results on nicorandil suggest the following: (1) The property of nicorandil as a resistive vessel dilator highly selective for vasculature originates in its mechanism of action as a K-channel activator. The nonunanimous effect of nicorandil on venous return is a result of the opposing actions as a capacitive (action as a nitrate) and a resistive vessel dilator. Nicorandil, with its hybrid nature, is advantageous over specific K-channel activators and classic nitrates in therapeutic implications.

Assessment of the effects of vasoactive intestinal peptide (VIP) on blood flow through and salivation of the dog salivary gland in comparison with those of secretin, glucagon and acetylcholine.

1. The vascular bed of the submandibular gland in situ was perfused with blood through the glandular artery at a constant pressure in anesthetized dogs. All drugs were administered intra-arterially. 2. Vasoactive intestinal peptide (VIP), secretin and acetylcholine produced a dose-dependent increase in blood flow through the artery (vasodilatation) but glucagon was almost ineffective. 3. Dose-blood flow response curves for VIP and secretin were parallel, and VIP was about 100 times as potent as secretin on a molar basis. Dose-blood flow response curves for acetylcholine were flatter than those for VIP and secretin. Acetylcholine was approximately as potent as secretin on a molar basis. 4. No tachyphylaxis developed to the vasodilator action of VIP. 5. The vasodilator responses to VIP and to electrical stimulation of the chordolingual nerve were scarcely modified by (-)-hyoscyamine in doses that fully antagonized the vasodilator response to acetylcholine. 6. VIP, secretin and glucagon were ineffective in eliciting salivary secretion. 7. The possibility that VIP is released from parasympathetic vasodilator nerves and mediates the atropine-resistant vasodilatation in the dog submandibular gland is discussed.

Differential effects of the calcium-antagonistic vasodilators, nifedipine and verapamil, on the tracheal musculature and vasculature of the dog.

In anaesthetized dogs the trachea in situ was perfused arterially with blood, and drugs were injected intraarterially. Isoprenaline and the calcium-antagonistic vasodilators, nifedipine and verapamil, increased tracheal blood flow and decreased resting tone of the trachea. Isoprenaline was equi-effective in producing tracheal vasodilatation and tracheal dilatation. The two calcium-antagonistic vasodilators were less effective tracheal dilators than tracheal vasodilators.

Pharmacological analysis of salivary and blood flow responses to histamine of the submandibular gland of the dog.

1 The submandibular gland in situ was perfused with blood through the glandular artery at constant pressure in anaesthetized dogs. Drugs were administered intra-arterially. 2 Histamine produced both salivation and an increase in blood flow, each response having an early and a late component. 3 Marked tachyphylaxis to histamine developed in both of the salivary responses but only in the late blood flow response to histamine. 4 The early and late salivary responses were abolished and the late blood flow response was diminished by infusion of tetrodotoxin in doses that abolished the salivary and blood flow responses to electrical stimulation of the chorda-lingual nerve. 5 The whole salivary response to histamine was abolished by infusion of (--)-hyoscyamine in doses that greatly antagonized the salivary and blood flow responses to acetylcholine, whereas the blood flow responses to histamine were scarcely modified. These doses of (--)-hyoscyamine abolished the salivary response to chorda-lingual nerve stimulation but left the blood flow response to it unaffected. 6 The salivary and blood flow responses to histamine were unaffected by infusion of hexamethonium in doses that almost abolished the salivary and blood flow responses to chorda-lingual nerve stimulation. 7 The whole salivary response to histamine was abolished and the late blood response to histamine was partially inhibited by the histamine H1-receptor antagonist, mepyramine, but not by the histamine H2-receptor antagonist, metiamide. 8 The early blood flow response to histamine was antagonized by both mepyramine and metiamide but mepyramine was far more effective than metiamide. 9 These results led to the following conclusions: (1) the whole salivary response and a part of the late blood flow response to histamine are due entirely to excitation of parasympathetic postganglionic neurones; (2) neuronal histamine receptors involved are exclusively of the H1-type; (3) histamine has no direct stimulant action on the glandular cells; (4) the early blood flow response and the remaining part of the late blood flow response to histamine result from the direct action on vascular smooth muscle in the glandular vascular bed; (5) vascular histamine receptors consist of H1- and H2-receptors.

Vasodilatation by prostaglandin F2alpha in the canine tongue through a parasympathetic mechanism.

1 The vascular bed of the tongue in situ was perfused with blood through the lingual arteries at a constant pressure in anaesthetized dogs. All drugs except for SQ 14,225 were administered intra-arterially.2 Prostaglandin F(2alpha) (PGF(2alpha)) produced a dose-dependent increase in blood flow through the lingual arteries (vasodilatation).3 Marked desensitization was observed on the vasodilator responses to repeated administration of PGF(2alpha).4 The vasodilator response to PGF(2alpha) was abolished by tetrodotoxin in doses that abolished the vasodilator response to electrical stimulation of the lingual nerve.5 The vasodilator response to PGF(2alpha) was not affected by hexamethonium in doses that almost abolished the vasodilator response to lingual nerve stimulation.6 The vasodilator responses to PGF(2alpha) and to lingual nerve stimulation were scarcely modified by (-)-hyoscyamine in doses that fully antagonized the vasodilator response to acetylcholine.7 Electrical stimulation of the vago-sympathetic trunk and noradrenaline produced a decrease in blood flow through the lingual arteries.8 These results indicate that the vasodilator response of the tongue to PGF(2alpha) is due exclusively to excitation of parasympathetic postganglionic neurones and that neuronal receptors involved are quite distinct from nicotinic receptors.9 Intravenous administration of SQ 14,225, an inhibitor of angiotensin I converting enzyme or kininase II, augmented the vasodilator responses to bradykinin and kallikrein but not that to lingual nerve stimulation.10 The results suggest that neither kallikrein nor*kinin (including bradykinin) is responsible for the parasympathetically induced vasodilatation in the tongue.

Pharmacological analysis of histamine receptors in musculature and vasculature of the dog trachea in situ.

1 The role of histamine H1- and H2-receptors in the musculature and vasculature of the dog trachea was investigated in the blood-perfused trachea in situ. 2 Histamine and acetylcholine caused increases in blood flow (tracheal, vasodilatation) and in intraluminal pressure (tracheal constriction) in a dose-dependent manner. Histamine was almost equipotent to acetylcholine in causing tracheal vasodilatation but was about 30 times less potent in causing tracheal constriction. 3 The histamine H2-receptor agonist, dimaprit, caused a dose-dependent increase in tracheal blood flow but failed to cause tracheal constriction. 4 The tracheal constriction produced by histamine was inhibited strongly by diphenhydramine but not modifed by metiamide. The tracheal vasodilatation produced by histamine was antagonized by both diphenhydramine and metiamide; diphenhydramine was more effective than metiamide. 5 It is concluded that in the tracheal musculature, histamine receptors are exclusively of the H1-type and mediate constriction, whereas in the tracheal vasculature, both histamine H1- and H2-receptors mediate vasodilatation but histamine H1-receptors are predominant.

A method for recording smooth muscle and vascular responses of the blood-perfused dog trachea in situ.

1 A method is described for measuring responses of dog tracheal musculature and vasculature in situ. 2 The upper two thirds of the trachea was perfused with blood through both cranial thyroid arteries at a constant pressure. The blood flow through the arteries was measured with an electromagnetic flowmeter. The response of the tracheal musculature was measured as a change in pressure in a water-filled cuff inserted into the trachea via the mouth. Drugs were injected close-arterially. 3 Acetylcholine produced dose-dependent increases in blood flow rate (vasodilatation) and in tracheal intraluminal pressure (tracheal constriction). These responses were antagonized by atropine. 4 Isoprenaline produced vasodilatation which was blocked by propranolol. Adrenaline and noradrenaline caused vasocontriction which was blocked by phentolamine. 5 All three catecholamines produced a decrease in tracheal intraluminal pressure (tracheal dilatation). The tracheal dilatation in response to adrenaline and noradrenaline was converted to constriction by propranolol. The tracheal constriction thus unmasked was abolished specifically by phentolamine. 6 From these results it is concluded that the tracheal musculature and vasculature contain muscarinic receptors, and excitatory alpha- and inhibitory beta-adrenoceptors. In the tracheal musculature beta-adrenoceptors predominate over alpha-adrenoceptors; the reverse is true in the tracheal vasculature.

Assessment of the selectivity of OPC-2009, a new beta2-adrenoceptor stimulatn, by the use of the blood-perfused trachea in situ and of the isolated blood-perfused papillary muscle of the dog.

1 The potency and selectivity of 5-(1-hydroxy-2-isopropylamino)butyl-8-hydroxy carbostyril hydrochloride hemihydrate (OPC-2009), a new beta(2)-adrenoceptor stimulant, was compared with those of isoprenaline, trimetoquinol and salbutamol by the use of blood-perfused tracheal preparations in situ and of blood-perfused papillary muscle preparations of the dog. All drugs were injected intra-arterially.2 All the four drugs decreased tracheal intraluminal pressure (tracheal relaxation) and increased tracheal blood flow in a dose-dependent manner. The four drugs produced a dose-dependent increase in developed tension of papillary muscles. In both preparations the duration of action of isoprenaline and salbutamol was short, whereas that of OPC-2009 and trimetoquinol was long. These effects were antagonized by propranolol.3 Dose-response curves to the four drugs for tracheal relaxation were almost parallel. OPC-2009 was 2.4 times more potent, and trimetoquinol and salbutamol were 2.2 and 6.2 times less potent than isoprenaline in causing tracheal relaxation.4 Dose-response curves to the four drugs for tracheal vasodilatation were also parallel. OPC-2009, trimetoquinol and salbutamol were 3.9, 6.7 and 23 times less potent than isoprenaline.5 Slopes of the dose-response curves to the four drugs for increased developed tension were not parallel; that of OPC-2009 was the least steep, whereas that of isoprenaline was the steepest. Trimetoquinol, salbutamol and OPC-2009 were about 18, 570 and 2400 times less potent than isoprenaline.6 Selectivity calculated from relative potencies indicate that OPC-2009 was about 6000 times, salbutamol about 92 times and trimetoquinol about 8.2 times more selective than isoprenaline for tracheal smooth muscle as compared to ventricular muscle.7 The high potency and selectivity of OPC-2009 for tracheal smooth muscle and its long duration of action suggest its potential usefulness for treatment of bronchial asthma.8 The present results are also compatible with the concept that beta(1)-adrenoceptors in cardiac muscle and beta(2)-adrenoceptors in tracheal and vascular smooth muscle can be distinguished. Furthermore, the results revealed that the beta-adrenoceptors mediating tracheal relaxation and vasodilatation may also be different.

Differential antagonism by Bay K 8644 of vasodilator effects of nifedipine, diltiazem, nicorandil and nitroglycerin in dog femoral circulation.

1. The modification by Bay K 8644 of the vasodilator effects of nifedipine, diltiazem, nicorandil and nitroglycerin was investigated in the femoral arterial bed of anaesthetized dogs. 2. The right femoral artery was cannulated and its arterial bed was perfused with autologous blood at a constant pressure slightly higher than the mean systemic arterial blood pressure. Bay K 8644 was infused intra-arterially (i.a.) and the 4 vasodilators were injected i.a. as bolus doses. 3. The vasodilator effects of nifedipine (0.3-10 nmol), diltiazem (0.01-1 mumol), nicorandil (0.1-10 mumol) and nitroglycerin (0.3-100 nmol) were all suppressed by infusions of Bay K 8644 (3-100 nmol min-1). 4. The dose-response curve of nifedipine was shifted parallel to the right by the infusion of Bay K 8644 and the dose-ratio was the greatest of the 4 drugs. 5. The dose-response curve of diltiazem was also shifted to the right by Bay K 8644. However, the dose-ratio was far smaller than that of nifedipine. 6. The vasodilator effect of nicorandil was not antagonized as much by Bay K 8644 as that of nitroglycerin. This less effective antagonism of nicorandil by Bay K 8644 can be explained if nicorandil, which although structurally a nitrate, can in addition cause relaxation of vascular smooth muscle by hyperpolarizing the membrane which would result in Bay K 8644 being less effective.

Differential antagonism of the negative inotropic effect of gentamicin by calcium ions, Bay K 8644 and isoprenaline in canine ventricular muscle: comparison with cobalt ions.

1. Gentamicin (10(-4)-10(-2) M) and Co2+ (10(-4)-10(-2) M) produced a decrease in developed tension of canine isolated ventricular muscles leading to near abolition at 10(-2) M. Their negative inotropic effects developed rapidly and wore off shortly after wash-out. 2. The concentration-negative inotropic effect curves for gentamicin were shifted to the right in a parallel manner by increasing external Ca2+, or by the presence of Bay K 8644 (10(-7)-10(-5) M) or isoprenaline (10(-7)-10(-5) M). IC50 values for gentamicin increased about 3-fold with about a 6 fold increase in external Ca2+. The Schild plot yielded a pA2 of 2.29 for Ca2+ and its slope was -1.17 (r = -0.79). 3. The concentration-negative inotropic effect curves for Co2+ were shifted to the right in a parallel manner by increasing external Ca2+, or by the presence of isoprenaline (10(-7)-10(-5) M). IC50 values for Co2+ increased about 5 fold with about a 6 fold increase in external Ca2+. The Schild plot gave a pA2 value of 2.60 for Ca2+ and its slope was -1.11 (r = -0.86). 4. The concentration-positive inotropic effect curves for Ca2+, which were computer-fitted to a logistic equation, gave 2.88 x 10(-3) M for EC50. This value was very close to the KCa calculated from pA2 values for Ca2+ based on antagonism between gentamicin or Co2+ and Ca2+ (5.13 x 10(-3) and 2.51 x 10(-3) M). 5. It is concluded that like Co2+, gentamicin molecules compete with Ca2+ for the same binding sites presumably located at the outer orifice of Ca-channels in the cardiac sarcolemma.

Interaction of potassium channel openers and blockers in canine atrial muscle.

1. The possibility that the interaction between potassium channel openers, e.g. cromakalim, pinacidil and nicorandil, and some potassium channel blockers involves a common site was investigated in canine atrial muscle. 2. Cromakalim, pinacidil and nicorandil produced a negative inotropic effect, their pD2 (-log EC50) values being 6.11 +/- 0.07, 5.37 +/- 0.09 and 4.55 +/- 0.07, respectively. 3. The potassium channel blockers, tetraethylammonium (TEA), tetrabutylammonium (TBA), 3,4-diaminopyridine (DAP), CsCl and BaCl2 all produced a positive inotropic effect. 4. The concentration-effect curves for the negative inotropic actions of pinacidil were shifted in a parallel way to the right by low concentrations of TEA, TBA or BaCl2. Maximum responses to pinacidil were depressed by higher concentrations of the blockers. An analysis of the non-competitive antagonism by TEA yielded pKA (-log KA) values of 4.00-4.05 for pinacidil. 5. The concentration-effect curves for cromakalim and nicorandil were shifted by TEA similarly to those for pinacidil, and a similar analysis yielded pKA values of 4.47-4.68 for cromakalim and 3.47-3.74 for nicorandil. 6. The KA values of cromakalim, pinacidil and nicorandil were about 10-30 times greater than their EC50 values, indicating that there are non-linear stimulus-effect relationships between the binding of the three potassium channel openers to their binding sites at potassium channels and their negative inotropic effects. 7. The dissociation constants for TEA could also be estimated from pA2 and pKB values for antagonizing competitively and non-competitively the negative inotropic effects of the three potassium channel openers; they were 3.47-3.89, and did not differ between the potassium channel openers. 8. The concentration-effect curves for the three potassium channel openers were not affected by DAP or CsCl. 9. These results suggest the following: (i) quaternary ammonium compounds like TEA and TBA antagonize the negative inotropic effect of cromakalim, pinacidil and nicorandil by binding to potassium channels, thus preventing binding of the channel openers to the same sites or closely related sites in canine right atrial muscles.

Nitroglycerin relaxes canine coronary arterial smooth muscle without reducing intracellular Ca2+ concentrations measured with fura-2.

1. Changes in cytoplasmic Ca2+ concentration ([Ca2+]1) were measured simultaneously with force by a microfluorometric method using a calcium indicator, fura-2, in canine coronary arterial smooth muscle cells. 2. Depolarization by high (30-90 mM) KCl-physiological salt solution (PSS) produced concentration-dependent increases in force and [Ca2+]i. 3. The KCl-induced increase in [Ca2+]i abolished by Ca2+-free conditions and almost abolished by verapamil 10-5 M, suggesting that it was entirely due to the increased Ca2+ influx through voltage-dependent Ca2+ channels. 4. The [Ca2+]i force relationship in the presence of verapamil was not distinguishable from that of control. 5. Nitroglycerin produced a concentration-dependent, reversible contraction of the coronary artery that had been contracted by high KCl-PSS, without reduction of the increased [Ca2+]i. 6. The KCl-induced increase in [Ca2+]i was not affected by nitroglycerin and in a presence of nitroglycerin it was abolished by 10-5 M verapamil suggesting that it was caused by the influx of extracellular Ca2+. 7. The [Ca2+]-force curve was shifted to the right by nitroglycerin. 8. It is likely that nitroglycerin relaxes the coronary arterial smooth muscle b reducing the amount of myosin light chain phosphorylation even in the presence of raised [Ca2+]i produced by increased Ca2+ influx following depolarization.