Shinichiro Kokubun

Modulation by intracellular ATP and cyclic AMP of the slow inward current in isolated single ventricular cells of the guinea‐pig.

Effects of ATP and of cyclic AMP on membrane current systems were investigated in isolated single ventricular cells from guinea‐pig hearts by applying the suction electrode method. The intracellular milieu was dialysed with various solutions which were perfused continuously through the suction pipette. The presence of ATP, cyclic AMP and EGTA in the perfusion solution kept the plateau phase of the action potential almost intact for as long as 30 min. With depolarizing voltage‐clamp pulses from holding potentials between ‐30 and ‐40 mV, the slow inward current (isi) was activated at potentials positive to ‐20 mV. The inactivation time course of isi was fitted by two exponential components in the potential range between ‐10 mV and +30 mV. By increasing ATP from 2 to 9.5 mM in the solution, the amplitude of isi was increased and the slow component of inactivation was accelerated. The steady‐state current‐voltage relationship (I‐V curve), exhibited a negative slope that became steeper after increasing the ATP concentration. The current was shifted towards the outward direction between ‐40 mV and ‐10 mV and became more inward between ‐10 mV and +40 mV. Increase of the cyclic AMP concentration from 30 to 60 microM also enhanced the amplitude of isi, but the negative slope in the steady‐state I‐V curve was unaffected. Assuming that the concentration of free Ca2+ in the cell was well buffered at a low level by the EGTA‐Ca buffer solution in the pipette, it was concluded that [ATP]i and [cyclic AMP]i exert a direct influence on membrane current systems of the ventricular cell.

Membrane currents in the rabbit atrioventricular node cell.

The rabbit A-V node was dissected into pieces (0.2×0.2×0.2 mm) smaller than its space constant of 692±96 μm (n=5). These small specimens showed spontaneous action potentials whose configurations were similar to those of large specimens before dissection. The membrane time constant was 21.5±1.5 ms (n=5).Voltage clamp experiments were performed on the above specimens using the two-microelectrode technique. On depolarization from the holding potential of −40 mV to various potential levels a transient inward current and delayed outward current were recorded. On repolarization an outward current tail was observed. The transient inward current was blocked by application of D600 (2×10−7 g/ml) but was insensitive to TTX (1×10−7 g/ml). The inward current was decreased by superfusion with Na- or Ca-free Tyrode solution. Thus, this current was classified as the slow inward current (is). When the K concentration in the Tyrode solution was varied, the reversal potential of the outward current tail changed as expected for a K electrode, indicating that the outward current was carried by K ions. On hyperpolarization slow activation of inward current was recorded. The reversal potential of this current was between −20 and −30 mV, which was analogous to hyperpolarization activated current,ih, in the S-A node. A contribution of sodium current (iNa) to the action potential was obviously demonstrated from an inhibitory effect of TTX on the upstroke of the anodal break excitation. The ionic selectivity of each current system is compared with analogous current systems in other cardiac tissues and a possible mechanism for the slow conduction in the A-V node is discussed.

A region of the muscarinic-gated atrial K+ channel critical for activation by G protein βγ subunits

Abstract Complementary DNAs encoding two types of inwardly rectifying K + channels, GIRK1 and IRK1, have been cloned from rat atrium and mouse macrophage, respectively. GIRK1 expressed in Xenopus oocytes was activated by acetylcholine when m2 muscarinic acetylcholine receptor was coexpressed. The acetylcholine-induced activation of GIRK1 was enhanced by coexpression with the G protein β1γ2 subunit but not the β1γ1 or a subunits. Deletion of the C-terminus of GIRK1 impaired the channel activation associated with the β1γ2 subunit. Moreover, replacement of the C-terminus of IRK1 with that of GIRK1 produced a chimera channel that was activated by the β1γ2 subunit, whereas intact IRK1 was not activated by the S1γ2 subunit. These findings define the C-terminus of GIRK1 as a regulatory region for the G protein βγ subunit.

Single anion-selective channel and its ion selectivity in the vascular smooth muscle cell

The properties of voltage-gated Cl channels of cultured smooth muscle cell prepared from embryonic rat aorta were studied. In the excised patch (inside-out configuration), we observed the activity of channels, opening and closing spontaneously, when the membrane potential was held at around 0 mV. The channels were active at a potential range between +10 and −10 mV. A step change of the membrane potential from the active potential range in either a positive or a negative direction closed the channel to an apparently inactivated state. The time course of this inactivation process became faster as the amplitude of the step change was increased. Returning the membrane potential to 0 mV allowed the channel to recover from the inactivated state. The channel had at least two open conductance states. Ca ions at the cytoplasmic face were not required for the activation of the channel. Adenosine nucleotides at the same side of the membrane had no effect on channel activity. The channels were selective to anions rather than cations, and they had a large single channel conductance of 340.5±20.4 pS in symmetrical 150 mM TEA-Cl. The reversal potential of the channel was shifted by −15.2±2.6 and 17.0±1.7 mV, when the Cl concentration at the intracellular side was changed to 75 mM or 300 mM, respectively. The permeability sequence of halides was I−>Br−>Cl−>F− (1.4∶1.3∶1.0∶0.7), whereas the conductance sequence was Cl−>Br−>F−>I− (1.00∶0.89∶0.86∶0.83). The internal dimension of the channel was estimated by measuring the permeability of various anions with different molecular cross section. We suggest that the smallest cross section of the channel pore is about 32Å2.

Haplotype Analysis of the Human Renin Gene and Essential Hypertension

Abstract—The human renin gene is an attractive candidate for involvement in the underlying cause of essential hypertension (EH). Despite extensive examination, the relation between the renin gene and hypertension remains unclear. The aims of the present study were to discover new genetic markers of EH and to investigate the relations between polymorphisms of the renin gene and EH in the Japanese. Using the polymerase chain reaction–single strand conformation polymorphism (PCR-SSCP) method, we isolated 3 novel variants of the renin gene; a single nucleotide polymorphism (SNP) in intron 4 (T+17int4G), a variable number of tandem repeats (VNTR) polymorphism in intron 7, and a missense mutation in exon 9 (G1051A). We performed an association study with these polymorphisms in 212 patients with EH and 209 age-matched normotensive (NT) subjects. The frequency of genotypes VNTR and T+17int4G did not differ significantly between the 2 groups, whereas the overall distribution of G1051A was significantly different between EH and NT. Haplotype analysis revealed that the overall distribution of haplotypes differed significantly between the EH and NT groups. PRA levels in patients with EH with the G/G genotype were significantly higher than in subjects with EH with G/A and A/A genotypes. These data suggest that the missense mutation in exon 9 may affect the enzymatic function of renin and consequently may be involved in the etiology of hypertension.

Blockade of Cl channels by organic and inorganic blockers in vascular smooth muscle cells

The effects of Cl channel blockers on large-conductance (LC-type) Cl channels of cultured vascular smooth muscle cells (VSMC) were studied in order to characterize the channel pharmacologically. Intracellular disulphonic stilbene derivatives, such as 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2′-disulphonic acid (SITS) inhibited Cl channel activity in a dose-dependent manner. An obvious inhibitory effect of DIDS in this condition was obtained at concentrations higher than 5 μM, and the complete inhibition was obtained at around 100 μM, which was almost 10 times less than the effective dose of SITS. The inhibitory effect of DIDS was reversible at a drug concentration of lower than 50 μM. Single-channel conductance decreased as the concentration of DIDS increased. This decrease in the conductance was a consequence of unresolved openings of the channel due to fast blocking and unblocking rates of the drug. The Cl channel was also obviously inhibited by extracellular DIDS at a concentration of 1 mM. In addition, in cell-attached patches, 500 μM DIDS applied extracellularly inhibited Cl channel activated by the application of polymyxin B. We also investigated the effect of Zn on Cl channels in VSMC. Intracellular Zn dose dependently and reversibly blocked the channel at the positive intracellular potential range, whereas at the negative intracellular potential range it did not block the channel activity. Results in this study suggest the diversity of Cl channels among various tissues.

Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension

Prostacyclin inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. The prostacyclin synthase (PGIS) gene is a candidate gene for cardiovascular disease. The purpose of this study was to locate possible mutations in the PGIS gene related to hypertension and cerebral infarction. Using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, we discovered a T to C transition at the +2 position of the splicing donor site of intron 9 in patients with essential hypertension (EH). In vitro expression analysis of an allelic minigene consisting of exons 8-10 revealed that the nucleotide transition causes skipping of exon 9. This in turn alters the translational reading frame of exon 10 and introduces a premature stop codon (TGA). A three-dimensional model shows that the splice site mutation produces a truncated protein with a deletion in the heme-binding region. This splice site mutation was found in only one subject in 200 EH patients and 200 healthy controls. Analysis of the patients family members revealed the mutation in two of the three siblings. The urinary excretion of prostacyclin metabolites in subjects with the mutation was significantly decreased. All subjects displaying the splice site mutation in the PGIS gene were hypertensive. In this study, we report a novel splicing mutation in the PGIS gene, which is associated with hypertension in a family. It is thought that this mechanism may involve in the pathophysiology of their hypertension.

A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K+ channels by G-protein βγ-subunits

To determine the interaction site(s) of ATP‐sensitive K+ (KATP) channels for G‐proteins, sulfonylurea receptor (SUR2A or SUR1) and pore‐forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS‐7. Intracellular application of the G‐protein βγ2‐subunits (Gβγ2) caused a reduction of ATP‐induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. Gβγ2 bound in vitro to both intracellular (loop‐NBD) and C‐terminal segments of SUR2A, each containing a nucleotide‐binding domain (NBD). Furthermore, a single amino acid substitution in the loop‐NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the Gβγ2‐dependent alteration of the channel activities. These findings provide evidence that Gβγ modulates KATP channels through a direct interaction with the loop‐NBD of SUR.

Contribution of P2-purinoceptors to neurogenic contraction of rat urinary bladder smooth muscle

1 The contribution of P2‐purinoceptors to neurogenic contraction was investigated in rat urinary bladder smooth muscle by measurement of isotonic tension. 2 Contraction of rat urinary bladder smooth muscle induced by electrical stimulation was decreased to 84.19±3.90% of the control (n= 16) in the presence of atropine (1 μm), which was further decreased to 38.80 ±2.75% of the control (n = 49) in the presence of both atropine and 10 μm α,β‐methylene adenosine 5′‐triphosphate (α,β‐Me ATP). 3 The contractile response induced by electrical stimulation in the presence of atropine and α,β‐Me ATP was decreased to 27.81 ±4.07% (n = 23) and 26.63 ±5.01% (n = 15) of the control, by the addition of 100 μm cibacron blue 3GA and 100 μm suramin, respectively. The application of 100 μm adenosine 5′‐0–2‐thiodiphosphate (ADPβS) in the presence of atropine and α,β‐Me ATP decreased the contractile response induced by electrical stimulations to 17.15 ±3.71% (n=15) of the control. 4 Pretreatment of muscle strips with 100 μm ADPβS significantly reduced the response to either 200 μm α,β‐methylene adenosine 5′‐diphosphate or 200 μm ADPβS. 5 Uridine 5′‐triphosphate (100 μm to 1 mM) concentration‐dependently contracted muscle strips, and this contraction was significantly antagonized by desensitization of P2‐receptors with α,β‐Me ATP (10 μm), and completely antagonized by pretreatment of muscle strips with both α,β‐Me ATP and ADPβS (100 μm). 6 Di(adenosine‐5′) tetraphosphate (30 and 100 μm) contracted muscle strips, whereas it failed to contract after desensitization of P2‐receptors. 7 It is suggested that about 20% of the neurogenic contraction of rat urinary bladder smooth muscle is mediated via ADPβS‐sensitive purinoceptors.

Subtypes of purinoceptors in rat and dog urinary bladder smooth muscles

1 Both adenosine and adenosine 5′‐triphosphate (ATP) (10 μm and 100 μm) relaxed 10 μm acetylcholine (ACh)‐induced contraction of rat bladder strips, which was completely antagonized by 100 μm 8‐(p‐sulphophenyl) theophylline. In dog bladder neither adenosine nor ATP inhibited ACh‐induced contraction. 2 P2x‐purinoceptor agonists contracted both rat and dog bladder strips with the potency order of α,β‐MeATP > ATP > ADP. 3 α,β‐MeADP (100 μm) induced a contraction of the rat bladder strip even after desensitization of P2x‐purinoceptors but failed to contract the dog bladder strip. 4 2‐MeSATP (1 μm to 300 μm) concentration‐dependently induced contraction of rat bladder strips; this contraction was significantly inhibited after desensitization of P2x‐purinoceptors. Cibacron blue 3GA (100 μm) antagonized the drug at concentrations lower than 30 μm, whereas it augmented the response to the drug at concentrations above 30 μm. 5 ADPβS (1 μm to 1 mm) concentration‐dependently induced contraction of rat bladder strips after desensitization of P2x‐purinoceptors; a contraction which was significantly antagonized by cibacron blue 3GA (100 μm). 6 It is concluded that three subtypes of purinoceptors, P1 (mediating relaxation), and P2x and another type of P2 (mediating contraction), exist in rat urinary bladder smooth muscle, whereas a single subtype of the receptor, P2x‐purinoceptor (mediating contraction) occurs in dog urinary bladder smooth muscle.