Effect of dipfluzine on L-type calcium current in guinea pig ventricular myocytes.

AIM: To study the effect of dipfluzine (Dip) on L-type calcium current in guinea pig ventricular myocytes. METHODS: Single myocytes were dissociated by enzymatic dissociation method. The current was recorded with the whole-cell configuration of the patch-clamp technique. RESULTS: Dip (0.3 - 30 micromol/L) reduced the voltage-dependently activated peak value of I(Ca-L) in a concentration-dependent manner. The characteristics of I-V relationship were not greatly altered by Dip, and the maximal activation voltage of I(Ca-L) in the presence of Dip was not different from that of control. Steady-state activation of I(Ca-L) was not affected markedly, and the half activation potential V(0.5)) and the slope factor (kappa) in the presence of Dip 3 micromol/L were not markedly different from those of the control. V(0.5) value was (-12.8 +/- 1.7) mV in the control and (-13.2 +/- 2.4) mV in the presence of Dip 3 micromol/L. The kappa value was (7.1 +/- 0.4) mV in the control and (7.5 +/- 0.5) mV in the presence of Dip 3 micromol/L (n = 7 cells from 3 hearts, P > 0.05). Dip 3 micromol/L markedly shifted the steady-state inactivation curve of I(Ca-L) to the left, and accelerated the voltage-dependent steady-state inactivation of calcium current. V(0.5) value was (-19.7 +/- 2.4) mV in the control and (-31 +/- 6) mV in the presence of Dip 3 micromol/L. The kappa value was (3.6 +/- 0.3) mV in the control and (1.8 +/- 0.2) mV in the presence of Dip 3 micromol/L (n = 4 cells from 2 hearts, P < 0.05). Dip 3 micromol/L markedly delayed half-recovery time of Ca2+ channel from inactivation from (40 +/- 11) to (288 +/- 63) ms (n = 4, P < 0.01). CONCLUSION: Dip mainly acts on the inactivated state of L-type calcium channel, accelerates the inactivation of calcium channel, and slows the recovery of calcium channel from inactivated state in guinea pig ventricular myocytes, through which the I(Ca-L) is inhibited.

[NADPH-diaphorase activity and Fos expression in brainstem nuclei involved in cardiovascular regulation following intracarotid injection of capsaicin].

The present study was undertaken to define whether intracarotid injection of capsaicin induces Fos expression associated with the activation of NOS-containing neurons in brainstem nuclei by combining the immunocytochemical method for Fos with NADPH-d histochemical technique for NOS. The results obtained are as follows: (1) Intracarotid injection of capsaicin caused a significant increase of Fos-like immunoreactive neurons in area postrema (AP), nucleus tractus solitarius (NTS), paragigantocellularis lateralis (PGL) and locus coeruleus (LC), without influence upon the neurons of raphe nuclei (RN) and periaqueductal gray (PAG). (2) NO-containing neurons in PGL and NTS and the double-labeled neurons in PGL were also increased significantly following intracarotid injection of capsaicin. Small numbers of NO-containing neurons were found in LC, but there was no change in the number of NO-containing neurons in RN and PAG. No NADPH-d histochemical activity could be found in AP. (3) The above responses to capsaicin were significantly inhibited by pretreatment with either a capsaicin receptor antagonist ruthenium red or a NMDA receptor antagonist MK-801. The above results indicate that intracarotid injection of capsaicin may activate the neurons in brainstem nuclei involved in cardiovascular regulation, and that NO only plays an indirect role in the modulation of the responses of brainstem nuclei to capsaicin. These effects of capsaicin are mediated by capsaicin receptors with involvement of glutamate.

Changes in heart rate, blood pressure and renal sympathetic nerve activity induced by microinjection of capsaicin into area postrema in rats.

The effects of capsaicin microinjection into area postrema (AP) on mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were investigated in 36 anesthetized Sprague-Dawley rats. The results obtained are as follows. (1) Following microinjection of capsaicin (10 micromol/L, 50 nl) into the AP, MAP, HR and RSNA were significantly increased from 12.34+/-0.53 kPa, 328.52+/-7.54 bpm and 100+/-0% to 15.17+/-0.25 kPa (P<0.001), 354.81+/-8.54 bpm (P<0.001) and 156.95+/-7.57% (P<0.001), respectively. (2) Ruthenium red (RR, 100 mmol/L, 0.2 ml, iv), a capsaicin receptor antagonist, significantly inhibited these effects of capsaicin. (3) Pretreatment with a NMDA receptor antagonist MK-801 (500 microgram/kg, 0.2 ml, iv) also reduced these effects of capsaicin. The above results indicate that microinjection of capsaicin into AP induces excitatory effects on MAP, HR and RSNA, which are mediated by capsaicin receptors with glutamate involvement.

[Increased expression of c-fos in the brainstem nuclei involved in cardiova scular regulation by capsaicin].

Effects of intracarotid injected capsaicin on the expression of Fos proto-oncogene in the brainstem nuclei involved in cardiovascular regulation were examined in 16 anesthetized rats with sinoaortic denervation. Only a few Fos-like protein immunoreactivity (FLI) neurons were found in the vehicle-control rats. Following intracarotid injection of capsaicin (10 micromol, 0.1 ml), the FLI neurons were markedly increased in nucleus paragigantocellularis lateralis (PGL), locus coeruleus (LC), area postrema (AP) and nucleus tractus solitarius (NTS), as compared with the results of vehicle-control rats, while the FLI neurons of raphe nuclei (RN) and periaqueductal gray (PAG) were not affected. The excitatory response to intracarotid injection of capsaicin was significantly inhibited by pretreatment with the vanilloid receptor (capsaicin receptor) antagonist ruthenium red (200 mmol, 0.1 ml). The results indicate that capsaicin may activate the neurons of the brainstem nuclei involved in cardiovascular regulation through vanilloid receptors, but does not exert any effects on PAG and RN.

[Effects of intracarotid injection of capsaicin on electrical activity of rostral ventrolateral medullary neurons in rats].

The effects of intracarotid injection of capsaicin on spontaneous electrical activity of nucleus paragigantocellularis lateralis (PGL) neurons in rostral ventrolateral medulla (RVLM) were examined in 35 anesthetized rats with sino-aortic denervation by using extracellular recording technique. The results obtained were as follows: (1) In response to intracarotid injection of capsaicin (10 mumol, 0.1 ml), MAP was increased from 10.74 +/- 0.13 to 12.56 +/- 0.21 kPa (P < 0.001), and HR from 374 +/- 4 to 395 +/- 5 bpm (P < 0.001). All of 30 PGL neurons recorded responded to intracarotid injection of capsaicin with an increase in spontaneous discharge rate from 12.6 +/- 0.7 to 20.9 +/- 1.1 spikes/s (P < 0.001); (2) In 10 units, the excitatory response of PGL neurons to intracarotid injection of capsaicin was significantly inhibited by pretreatment with the vanilloid receptor (capsaicin receptor) antagonist ruthenium red (200 mmol, 0.1 ml). These results suggest that capsaicin may stimulate the vanilloid receptor of neurons in RVLM, thereby resulting in activation of PGL neurons.

[Inhibitory effects of nitric oxide on glutamate-induced neuronal activity of CA1 area in rat hippocampal slices].

Using extracellular recording technique, the effects of L-arginine (L-arg), SIN-1 and N-nitro-L-arginine (L-NNA) on glutamate-induced discharge of neurons in CA1 area of hippocampal slices were examined to define the role of L-arg:NO pathway in glutamate-induced discharge of hippocampal neurons and its possible underlying mechanism. The results obtained are as follows. (1) In response to the application of glutamate (0.5 mmol/L) into the superfusate for 1 min, the discharge rate of 12 neurons was increased markedly in an epileptiform pattern. (2) The increased discharge induced by glutamate (0.5 mmol/L) in 10 neurons was suppressed significantly by application of L-arg (10 mmol/L) into the superfusate for 2 min. (3) The glutamate-induced increase of discharge in 12 neurons was decreased markedly by superfusing the brain slice with NO donor SIN-1 (5 mmol/L) for 1 min. (4) As the discharge rate of 12 neurons was increased by pretreatment with glutamate (0.5 mmol/L), application of L-NNA (0.15 mmol/L) into superfusate for 2 min might further augment the discharge intensively and in some case eventually led to abrupt suppression of the discharge. Taken together, it is likely that glutamate binding with NMDA receptors in hippocampal neurons not only induces an increase in discharge, but also activates the L-arg: NO pathway to generate NO responsible for neuroprotection via negative feedback mechanisms.

[Effects of no precursor and donor on neuronal activity of rat hippocampal slices].

Using extracellular recording technique, the effects of L-arginine (L-arg), N-nitro-L-arginine (L-NNA), SIN-1 and methylene blue (MB) on spontaneous discharges of neurons in CA1 area of hippocampal slices were examined to determine the role of L-arg: NO pathway and the possible underlying mechanism. The results were as follows: (1) In response to the application of L-arg (1 mmol/L) into the superfusate for 2 min, spontaneous discharge rate (SDR) of 42/54 (77.8%) neurons was decreased significantly, while that of 12/54 (22.2%) neurons showed no change. Following the application of L-NNA (0.15 mmol/L) into the superfusate for 2 min, SDR of 25/29 (86.2%) neurons was increased markedly and that of 4/29 (13.8%) neurons was not affected. The effect of L-NNA might be reversed by pretreatment with L-arg. (2) With application of NO donor SIN-1 (5 mmol/L), SDR of 25 (100%) neurons was decreased in a dose-dependent manner. (3) After superfusing the brain slice with guanylate cyclase inhibitor, MB (3 mumol/L) for 30 min, SDR of 10 units showed significant increase as compared with control. However, MB failed to abolish the effect of L-arg on hippocampal neurons. Taken together, it is likely that NO is released during the resting state of hippocampal neurons and may inhibit the activity of hippocampus, an effect not mediated by the action of guanylate cyclase.