Premium
Modulation of guinea‐pig cardiac L‐type calcium current by nitric oxide synthase inhibitors
Author(s) -
Gallo Maria Pia,
Ghigo Dario,
Bosia Amalia,
Alloatti Giuseppe,
Costamagna Costanzo,
Penna Claudia,
Levi Renzo C.
Publication year - 1998
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1998.639bv.x
Subject(s) - nitric oxide synthase , omega n methylarginine , nitric oxide , western blot , extracellular , arginine , chemistry , stimulation , patch clamp , electrophysiology , biology , endocrinology , medicine , biochemistry , amino acid , gene
1 Electrophysiological (whole‐cell clamp) techniques were used to study the effect of NO synthase (NOS) inhibitors on guinea‐pig ventricular calcium current ( I Ca ), and biochemical measurements (Western blot and citrulline synthesis) were made to investigate the possible mechanisms of action. 2 The two NOS inhibitors, N G ‐monomethyl‐L‐arginine (L‐NMMA, 1 mM) and N G ‐nitro‐L‐arginine (L‐NNA, 1 mM), induced a rapid increase in I Ca when applied to the external solution. D‐NMMA (1 mM), the stereoisomer of L‐NMMA, which has no effect on NOS, did not enhance I Ca . 3 Western blot experiments gave no indication of the presence of inducible NOS protein (iNOS) in our cell preparation, neither immediately after dissociation nor after more than 24 h. Statistically, there was no significant difference between electrophysiological experiments performed on freshly dissociated cells and experiments performed the next day. Moreover cells prepared and kept in the presence of dexamethasone (3 μM), to inhibit the expression of iNOS, gave the same response to L‐NMMA as control cells. 4 The stimulatory effect of L‐NMMA (1 mM) on basal I Ca was reversed by competition with higher doses (5 mM) of externally applied L‐arginine, the natural substrate of NOS. The effect of L‐NMMA was also eliminated by L‐arginine in the patch pipette solution. 5 Intracellular perfusion with GDPβS (0.5 mM), which stabilizes the G‐proteins in the inactive state, did not affect the L‐NMMA‐induced stimulation of I Ca . 6 Carbachol (1 μM) reduced the I Ca previously stimulated by L‐NMMA, and intracellular cGMP (10 μM) prevented L‐NMMA enhancement. 7 Simultaneous treatment with L‐NMMA and isoprenaline (1 μM) induced a non‐cumulative enhancement of I Ca that could not be reversed by carbachol (1 μM). 8 NO synthesis, measured by the formation of [ 3 H]citrulline from L‐[ 3 H]arginine during a 15 min incubation, showed a relatively high basal NO production, which was inhibited by L‐NMMA but not affected by carbachol. 9 These results suggest that inhibitors of NOS are able to modulate the basal ventricular I Ca in the absence of a receptor‐mediated pathway, and that NO might be required for the muscarinic reduction of I Ca under isoprenaline stimulation, even if NO production is not directly controlled by the muscarinic pathway.