Johanna Rupp

Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle

SummaryThe effects of 5-hydroxytryptamine (5-HT) on force of contraction (FC), action potential (AP) and calcium current (ICa) were studied in human right atrial and left ventricular heart muscle. 5-HT exerted a concentration-dependent increase in FC in multicellular atrial preparations; the EC50 was approximately 3 × 10−7 mol/l. Maximal increases in FC (252±58% of control values; means ± SEM, n=6) were obtained at 5-HT 10−5 mol/l. At this concentration, ICa was increased four- to sevenfold in enzymatically isolated atrial myocytes. In contrast, ventricular preparations did not respond to 5-HT; FC, AP and ICa remained unaffected. In the same preparations, FC was increased by isoprenaline three- to fourfold. These results confirm the observation that 5-HT induces a positive inotropic effect in the human atrium, possibly mediated by activation of the adenylyl cyclase — cyclic AMP system. Our study demonstrates, however, the complete lack of functional 5-HT receptors, with respect to changes in FC, in the human ventricle. Since the positive inotropic effect of 5-HT in the human heart is obviously restricted to the atrium, our findings question the concept of developing 5-HT receptor agonists for the treatment of heart failure.

The ineffectiveness of the NO‐cyclic GMP signaling pathway in the atrial myocardium

1 This study was performed to determine whether nitric oxide (NO) has direct effects on force of contraction (Fc) in atrial myocardium from rats, rabbits, guinea‐pigs, frogs, and man. 2 Glyceryl trinitrate, isosorbide dinitrate, 3‐morpholino‐sydnonimine hydrochloride (SIN‐1), and S‐nitroso‐N‐acetylpenicillamine (SNAP) did not signficantly reduce Fc in the various preparations investigated, either given alone or after stimulation of α‐ or β‐adrenoceptors. 3 SNAP did not change the time course of contractions in rat, guinea‐pig and human preparations. 4 8‐Bromo‐guanosine‐3′:5′‐cyclic monophosphate (8‐Br‐cyclic GMP) produced a negative inotropic effect in rat, guinea‐pig and human atrial preparations and shortened time to peak tension and relaxation time in human preparations. 5 High K+ (85 mmol 1−1‐induced contracture in rat heart muscle was reduced by 8‐Br‐cyclic GMP but not by SIN‐1. 6 N‐monomethyl‐L‐arginine (l‐NMMA), an inhibitor of NO synthase, failed to influence muscarinic effects on Fc or frequency from rat and guinea‐pig hearts. 7 We conclude that NO, under the experimental conditions described here, has no direct effects on the heart, although cyclic GMP may be involved in the regulation of myocardial contraction.

Adrenoceptor-mediated changes of action potential and force of contraction in human isolated ventricular heart muscle.

1 The effects of α‐adrenoceptor stimulation on the action potential and force of contraction were investigated in human isolated ventricular heart muscle and compared with those of β‐adrenoceptor Stimulation. 2 The maximal stimulation by isoprenaline of β‐adrenoceptors produced large changes in the force of contraction, which were accompanied by moderate increases in the height of the action potential. The maximal inotropic effect produced by stimulation of α‐adrenoceptors with phenylephrine, in the presence of propranolol (1 μmol l−1) was much smaller (about 10% of that seen in response to β‐adrenoceptor stimulation), and no significant changes of the action potential configuration were observed. 3 The effects of noradrenaline and adrenaline on the force of contraction were not affected by prazosin. 4 It is concluded that the adrenoceptor‐mediated changes of the force of contraction (in the presence of either noradrenaline or adrenaline) in the human ventricle are due virtually exclusively to the stimulation of β‐adrenoceptors.

L-type calcium channel activity in human atrial myocytes as influenced by 5-HT

Summary5-Hydroxytryptamine (10 μmol/l; 5-HT) exerted a positive inotropic effect associated with an increase in the Ca2+ current (ICa) in the human right atrium. For detailed analysis, L-type Ca2+ channel currents were recorded from cell-attached patches using 100 mmol/l Ba2+ as charge carrier. Ca2+ channel activity was identified, first, by burstlike inwardly directed currents and, second, by the appearance of long channel openings promoted by Bay K 8644 (1 μmol/l) upon repetitive depolarizations from − 80 to 0 mV The unitary conductance of the Ca2+ channel amounted to 25.8 pS. During superfusion with 5-HT, ensemble averaged (mean) current was enhanced by about 60%. The increase in mean current was brought about by an increase in the channel availability, defined as the ratio of sweeps containing Ca2+ channel activity to the total number of depolarizations. The open probability of a single Ca2+ channel within a sweep with channel activity, unitary conductance, mean open and mean shut times of the channel, however, remained unaffected during superfusion with 5-HT (n = 10). The 5-HT-induced increase in macroscopic ICa in the human atrium can therefore be explained by an enhanced availability of Ca2+ channels to open upon depolarization. The observed changes in gating properties of the human Ca2+ channel by 5-HT are very similar to those which are known from isoprenaline-induced CAMP-dependent phosphorylation of the Ca2+ channel protein in other tissues.

Functional role of cholinoceptors and purinoceptors in human isolated atrial and ventricular heart muscle

1 The effects of cholinergic and purinergic stimulation on action potential, force of contraction and 86Rb efflux were investigated in human atrial and ventricular heart muscle. 2 In atrial heart muscle, carbachol and (−)‐N6‐(R‐phenyl‐isopropyl)‐adenosine (R‐PIA) and 5′‐(N‐ethyl)‐carboxamido‐adenosine (NECA) evoked transient decreases of action potential duration and force of contraction; the steady‐state effects on force of contraction were virtually identical to control values. In the presence of propranolol, steady‐state values after carbachol, R‐PIA or NECA amounted to about 50% of control values. 3 In ventricular heart muscle, carbachol, NECA and R‐PIA did not significantly affect the action potential configuration or force of contraction. 4 Carbachol, NECA and R‐PIA induced a maintained depression of the positive inotropic response to isoprenaline in both atrial and ventricular heart muscle. 5 The rate constant of 86Rb efflux was slightly increased by carbachol, NECA and R‐PIA in atrial (10–20%) but not in ventricular heart muscle. 6 In the presence of isoprenaline, carbachol, NECA and R‐PIA did not significantly affect the rate constant of 86Rb efflux in both atrial and ventricular heart muscle. Isoprenaline alone increased the rate constant of 86Rb by about 25% in both tissues.

Proton conductance of human transient receptor potential–vanilloid type-1 expressed in oocytes of Xenopus laevis and in Chinese hamster ovary cells

Transient receptor potential-vanilloid type-1 (TRPV1) is a ligand-gated cation channel with preference for divalent cations, especially Ca(2+) (sequence of conductances: Ca(2+)>Mg(2+)>Na(+) approximately/= K(+) approximately/= Cs(+)). In the present study, the two-electrode voltage-clamp technique was used on oocytes of Xenopus laevis expressing TRPV1 to evaluate whether human TRPV1 also conducts protons. In medium devoid of K(+), Na(+), Mg(2+), and Ca(2+), capsaicin 1 microM induced a significant inward current (62% of the current in physiological medium). The effects of capsaicin were abolished in the presence of capsazepine 3 microM. The capsaicin-induced currents in medium devoid of Na(+), K(+), Mg(2+), and Ca(2+) were dependent on pH, causing larger inward currents and less negative reversal potentials at low pH and vice versa. The same current was also demonstrated in Chinese hamster ovary cells expressing human TRPV1. We conclude that TRPV1 conducts protons, in addition to Na(+), K(+), Mg(2+), and Ca(2+). The proton conductance may help to initiate action potentials and to translocate H(+) dependent on TRPV1 activation and membrane potential.

Barnidipine block of L-type Ca2+ channel currents in rat ventricular cardiomyocytes

The effects of barnidipine and nifedipine on L‐type Ca2+ current (ICa(L)) were investigated in ventricular cardiomyocytes from rats. Both barnidipine and nifedipine reduced ICa(L) in a concentration and voltage dependent manner; the EC50 were 80 and 130 nM at a holding potential of −80 mV, respectively, and 18 and 6 nM at −40 mV, respectively. Both drugs induced a leftward shift of the steady‐state inactivation curve of ICa(L). Using a twin pulse protocol, the relationships between the amount of block of ICa(L) by either drug, seen during the second pulse, and the length of the first pulse were described by monoexponential functions reflecting onset of block, dependent on drug concentration. The onset of block by barnidipine was three times faster than that by nifedipine. With both drugs, recovery of ICa(L) was 50 times slower than under control conditions and described by monoexponential functions reflecting offset of block (independent of drug concentration). The offset of block with barnidipine was three times slower than that with nifedipine. The time constants of block and unblock of ICa(L) by both drugs were used to calculate binding and unbinding and to predict their effects at two frequencies. It is suggested that barnidipine exhibits a higher affinity to the inactivated Ca2+ channel state as compared to nifedipine.

Induced arginine transport via cationic amino acid transporter-1 is necessary for human T-cell proliferation.

Availability of the semiessential amino acid arginine is fundamental for the efficient function of human T lymphocytes. Tumor‐associated arginine deprivation, mainly induced by myeloid‐derived suppressor cells, is a central mechanism of tumor immune escape from T‐cell‐mediated antitumor immune responses. We thus assumed that transmembranous transport of arginine must be crucial for T‐cell function and studied which transporters are responsible for arginine influx into primary human T lymphocytes. Here, we show that activation via CD3 and CD28 induces arginine transport into primary human T cells. Both naïve and memory CD4+ T cells as well as CD8+ T cells specifically upregulated the human cationic amino acid transporter‐1 (hCAT‐1), with an enhanced and persistent expression under arginine starvation. When hCAT‐1 induction was suppressed via siRNA transfection, arginine uptake, and cellular proliferation were impaired. In summary, our results demonstrate that hCAT‐1 is a key component of efficient T‐cell activation and a novel potential target structure to modulate adaptive immune responses in tumor immunity or inflammation.

The cAMP Pathway Sensitizes VR1 Expressed in Oocytes from Xenopus laevis and in CHO Cells

The vanilloid receptor 1 (VR1) is a heat-activated cation channel which also responds to capsaicin and other chemical stimuli. Protein kinase C has a stimulatory effect on VR1 activity, either alone or after activation with capsaicin. The influence of the cAMP-signaling pathway on the effects of capsaicin is controversial. To clarify this, the actions of capsaicin and the modulatory effects of forskolin, pCPT-cAMP, and isobutylmethylxanthine were studied in Xenopus laevis oocytes expressing rat VR1 and in CHO cells expressing human VR1. Capsaicin activated the VR1 channel and increased the intracellular calcium concentration. The effects of capsaicin were enhanced by forskolin, pCPT-cAMP, and isobutylmethylxanthine. A modulatory function of the cAMP system on VR1 activation could, therefore, modulate heat sensation and pain.

Hypomorphic variants of cationic amino acid transporter 3 in males with autism spectrum disorders

Cationic amino acid transporters (CATs) mediate the entry of L-type cationic amino acids (arginine, ornithine and lysine) into the cells including neurons. CAT-3, encoded by the SLC7A3 gene on chromosome X, is one of the three CATs present in the human genome, with selective expression in brain. SLC7A3 is highly intolerant to variation in humans, as attested by the low frequency of deleterious variants in available databases, but the impact on variants in this gene in humans remains undefined. In this study, we identified a missense variant in SLC7A3, encoding the CAT-3 cationic amino acid transporter, on chromosome X by exome sequencing in two brothers with autism spectrum disorder (ASD). We then sequenced the SLC7A3 coding sequence in 148 male patients with ASD and identified three additional rare missense variants in unrelated patients. Functional analyses of the mutant transporters showed that two of the four identified variants cause severe or moderate loss of CAT-3 function due to altered protein stability or abnormal trafficking to the plasma membrane. The patient with the most deleterious SLC7A3 variant had high-functioning autism and epilepsy, and also carries a de novo 16p11.2 duplication possibly contributing to his phenotype. This study shows that rare hypomorphic variants of SLC7A3 exist in male individuals and suggest that SLC7A3 variants possibly contribute to the etiology of ASD in male subjects in association with other genetic factors.