The present study was carried out to characterize the functional properties of spinomesencephalic tract (SMT) neurons in the lumbar spinal cord of urethane anesthetized rats. Extracellular single unit recordings were made from neurons antidromically activated by stimulation of the midbrain area, including the deep layers of superior colliculus, periaqueductal gray and midbrain reticular formation. Recording sites were located in laminae I-VII of spinal cord segments of L2-L5. Receptive field properties and responses to calibrated mechanical stimulation were studied in 78 SMT cells. Mean conduction velocity of SMT neurons was $19.1{\pm}1.04\;m/sec$. SMT units were classified according to their response profiles into four groups: wide dynamic range (58%), deep/tap (23%), high threshold (9%) and low threshold (3%). A simple excitatory receptive field was found for most SMT neurons recorded in superficial dorsal horn (SDH). Large complex inhibitory and/or excitatory receptive fields were found for cells in lateral reticulated area which usually showed long after-discharge. Most of SMT cells received inputs from $A{\delta}$ and C afferent fiber types. These results suggest that sensory neurons in the rat SMT may have different functional roles according to their location in the spinal cord in integrating and processing sensory inputs including noxious mechanical stimuli.

The present study was undertaken to elucidate the desensitization of cutaneous receptors and the conduction block of the afferent nerves induced by direct application of allyl isotheocyanate (mustard oil) to the receptive field (RF) or onto the afferent nerve, respectively. Dorsal horn cell responses to mechanical stimulations of RF were completely suppressed when mustard oil was applied to either the afferent nerve or the whole area of RF. C-fiber responses of dorsal horn cells were more susceptive to mustard oil than A-fiber activities. This was confirmed by the experiment in which the compound action potentials recorded from rat tibial nerve before and after topical application of mustard oil were compared. The higher the concentration of mustard oil and the longer the application time, the more powerful desensitization or conduction block was induced. From the results of the present study, it is suggested that the desensitization of the afferent fiber and sensory receptors induced by mustard oil results mainly from the conduction block of C-fiber in the primary afferent nerve.

The discharge patterns and peripheral nerve inputs to cardiovascular neurons were investigated in rostral ventrolateral medulla (RVLM) and raphe nucleus of cats. The data from the two were compared to determine their roles in cardiovascular regulation and the endogenous analgesic system. Animals were anesthetized with ${\alpha}-chloralose$ and single cell activities were recorded by carbon-filament microelectrode and their relationships with cardiovascular activity were analyzed. In RVLM area, a total of thirty-three cells were identified as cardiovascular neurons. During one cardiac cycle, the mean discharge rate of the neurons was $1.96{\pm}0.29$ and the peak activity was observed 45 ms after the systolic peak of arterial blood pressure. Thirteen cells could be activated antidromically by stimulation of the the $T_2$ intermediolateral nucleus. Forty-three raphe neurons were identified as cardiovascular neurons whose mean discharge rate during one cardiac cycle was $1.02{\pm}0.12$. None of these cells could be activated antidromically. Study of the interval time histogram of RVLM neurons revealed that the time to the first peak was $128{\pm}20.0\;ms$, being shorter than the period of a cardiac cycle. The same parameter found from the raphe neurons was $481{\pm}67.2\;ms$, which was much longer than the cardiac cycle length. Of seventeen RVLM neurons examined ten received only the peripheral $A{\delta}-afferent$ inputs, whereas six RVLM neurons received both $A{\delta}-$ and C-inputs; the remaining one cell received an inhibitory peripheral C-input. In contrast, nine of eleven raphe neurons were found to receive $A{\delta}-inputs$ only. We conclude that the main output of cardiovascular regulatory influences are mediated through the RVLM neurons. The cardiovascular neurons in the raphe nucleus appear to serve as interneurons transferring cardiovascular afferent information to the raphespinal neurons mediating the endogenous analgesic mechanisms.

The whole-cell mode of the patch clamp technique was used to study $Ca^{2+}-activated\;Cl^-\;current$ $(I_{Cl_{Ca}})$ in gastric antral myocytes. Extracellular application of caffeine evoked $Ca^{2+}-activated\;current$. In order to isolate the chloride current from background current, all known systems were blocked with specific blockers. The current-voltage relationship of caffeine-induced current showed outward rectification and it reversed at around $E_{Cl^-}$. The shift of reversal potential upon the alteration of external and internal chloride concentrations was well fitted with results which were calculated by the Nernst equation. Extracellular addition of N-phenylanthranilic acid and niflumic acid which are known anion channel blockers abolished the caffeine induced current. Intracellular application of a high concentration of EGTA also abolished this current. Application of c-AMP, c-GMP, heparin, or $AIF^-_4$ made no remarkable changes to this current. Sodium replacement with the impermeable cation N-methylglucamine or with $Cd^{2+}$ rarely affected this current. From the above results it is suggested that the caffeine induced current was a $Cl^-$ current and it was activated by intracellular $Ca^{2+}$.

Intracellular free $Ca^{2+}$ contributes to regulation of various events occurring in vascular smooth muscle cells. One of these events is modulating the membrane iou currents. Single smooth muscle cells were isolated from rabbit mesenteric artery. Three kinds of $Ca^{2+}-activated\;current$ were studied with the patch clamp method. $Ca^{2+}-activated\;K^+\;current$ with a large oscillation was recorded in the depolarized potential range. The single channel conductance of this current was about 250 pS. It was abolished by replacing intracellular $K^+\;with\;Cs^+$. A $Ca^{2+}-activated$ nonselective cation current was observed in both the depolarized and hyperpolarized potential ranges. And it was blocked by replacement of extracellular $Na^+$ with N-methylglucamine (NMG) or extracellular application of $Cd^{2+}$. $Ca^{2+}-activated\;Cl^-\;current$ was revealed in the whole voltage range and was blocked by niflumic acid. These results indicate that at least three kinds of $Ca^{2+}-activated$ ionic currents exist in smooth muscle cells from rabbit superior mesenteric artery.

The contraction of rabbit basilar artery was examined as a function of changes in the $Na^+$ electrochemical gradient in order to determine the contribution of $Na^+/Ca^{2+}$ exchange to the modulation of contractility. Ouabain $(10^{-5}\;M)$ or $K^+-free$ Tyrode solution caused an increase in tonic tension even in the presence of a $Ca^{2+}$ channel blocker $(10^{-6}\;M\;verapamil)$ and an ${\alpha}-receptor$ blocker $(10^{-5}\;M\;phentolamine)$. After treatment with ouabain $(10^{-5}\;M)$, contractions were augmented by reduction of external $Na^+$ concentration. The longer the treatment with ouabain $(10^{-5}\;M)$ was, the larger the amplitude of $Na^+-free$ contracture was. $Na^+-free$ contracture wag induced by either substitution of equimolar Tris for $Na^+$ or substitution of equimolar $Li^+\;for\;Na^+$. The competition between $Na^+\;and\;Ca^{2+}$ for the $Na^+/Ca^{2+}$ exchange carrier would exist, because it was observed that contractility was dependent on the $Na^+$ electrochemical gradient or the extracellular $Ca^{2+}$ concentration (2 mM, 4 mM). Ryanodine $(10^{-7}\;M)$, the blocker of intracellular $Ca^{2+}$ release from the sarcoplasmic reticulum, did not suppress the development of $Na^+-free$ contracture. The contractile response to norepinephrine $(10^{-6}\;M)$ was augmented by reducing the extracellular $Na^+$ concentration. The relaxation rate from caffeine-induced contraction was dependent on the extracellular $Na^+$ concentration (0 mM, 140 mM). From the above results, it could be suggested that $Na^+/Ca^{2+}$ exchange can move $Ca^{2+}$ either into or out of rabbit basilar arterial smooth muscle. $Ca^{2+}$ entry or extrusion is dependent upon the $Na^+$ electrochemical gradient. $Na^+/Ca^{2+}$ exchange plays a significant role in the regulation of contractility in rabbit basilar arterial smooth muscle.

The effect of extracellular and intracellular pH on vascular tone and $^{45}Ca$ uptake were investigated in aortic strips and dispersed single aortic smooth muscle cells of spontaneously hypertensive rats (SHR) and aged-matched Wistar-Kyoto rats (WKY). The contraction produced by a change of extracellular pH (pHo) in the range of $6.5{\sim}8.3$ was estimated by comparison with the level of vascular tone at pH 7.4. Contraction was induced below pHo 6.5 in WKY, pHo 7.1 in SHR, and over pHo 8.0 on both strains. The amplitude of contraction induced by high pHo (over pHo 7.7) was similar in SHR and WKY, but that induced by low pHo (below pHo 7.1) in SHR was greater than that in WKY. Either high pHo- or low pHo-induced contractions in WKY and SHR were not induced in the Ca-free Tyrode's solution and were induced by the addition of Ca. $^{45}Ca$ uptake increased progressively as pHo was increased from 6.8 to 8.1 in the single aortic smooth muscle cells of WKY and SHR. $NH_4Cl$ induced a gradually developing contraction in a dose-dependent manner $(5\;mM{\sim}30\;mM)$ and the removal of $NH_4Cl$ induced transient contraction was followed by profound relaxation in the aortic rings of both strains. The contractions induced by $NH_4Cl$ or by the removal of $NH_4Cl$ in SHR were significantly greater than that in WKY. These contractions were not induced in Ca-free Tyrode's solution. $^{45}Ca$ uptake was increased by $NH_4Cl$ (20 mM) and was not changed by the removal of $NH_4Cl$ (20 mM) in the aortic strips of WKY and SHR. As a summary of above results, the vascular tone of SHR was more sensitive to the change pHi and pHo than that of WKY. The contractions induced by change of extracellular or intracellular pH depended on extracellular Ca in the aorta of SHR nnd WKY. However, the Ca uptake was in accord with the changes of contraction but increase in contraction by low pH was not accompanied by an increase in Ca uptake in both strains.

Endothelium-derived relaxing factor (EDRF) activates guanylate cyclase which mediates the formation of cGMP from GTP in vascular smooth muscle. It is well known that endothelium-dependent relaxation is impaired in spontaneously hypertensive rats (SHR). However, it is still unknown whether the impaired endothelium-dependent relaxation in SHR results from the reduced release of EDRF or from the decrease of vascular response to EDRF. We investigated the effects of cGMP on the contractility and Ca movement in the aorta of SHR and Wistar-Kyoto rats (WKY). The amplitude of the endothelium-dependent relaxation to actylcholine (ACh) was significantly less in SHR than in WKY. L-arginine $(10^{-3}M)$ did not increase endothelium-dependent relaxation in both strains. Sodium nitroprusside (SNP), an activator of guanylate cyclase, relaxed the 40 mM $K^+-induced$ contraction in a dose-dependent manner $(10^{-10}{\sim}10^{-6}\;M)$ in the endothelium-rubbed aortic strips of both strains. However, there was no significant difference in these relaxations between WKY and SHR. 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP), a cell membrane-permeable derivative of cGMP relaxed the 40 mM $K^+-induced$ contraction in a dose-dependent manner $(10^{-6}{\sim}10^{-4}\;M)$ in the endothelium-rubbed aortic strips of both strains. Also norepinephrine $(10^{-6}\;M)-induced$ contractions in normal and Ca-free Tyrode's solution were suppressed by the pretreatment with 8-Br-cGMP $(10^{-4}\;M)$ in either strain. However, the amplitudes of suppression induced by 8-Br-cGMP were greater in SHR than that in WKY. Basal $^{45}Ca$ uptake and 40mM $K^+-stimulated\;^{45}Ca$ uptake were not suppressed by pretreatment with 8-Br-cGMP $(10^{-4}\;M)$ in single aortic smooth muscle cells of both SHR and WKY. From the above results, it is suggested that cGMP decreases Ca sensitivity in vascular smooth muscle cells and that the impaired endothelium-dependent relaxation in the aortic strips of SHR is not the result of a reduced vascular response to EDRF.

Captopril, an inhibitor of angiotensin converting enzyme, is also known to inhibit the degradation of bradykinin. We examined the effects of intracerebroventricular (ICV) captopril on the central pressor response to bradykinin in normotensive, 2-kidney, 1 clip Goldblatt (GHR) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Captopril (1 mg) and bradykinin (5 nmol) were administered into the right lateral cerebral ventricle, and blood pressure and heart rate were continuously monitored throughout the experiment. ICV captopril alone did not affect the blood pressure within 10 minutes but it significantly augmented the central pressor response to bradykinin in GHR. On the contrary, captopril was without effect on the pressor response to bradykinin in normotensive and DOCA-salt rats. These findings indicate that endogenous kinins are not critical in regulating arterial pressure in normotensive and DOCA hypertensive rats. However, in GHR, an enhanced activity of the brain kallikrein-kinin system in maintaining the high blood pressure is suggested.

The present study was aimed to determine if endogenous L-arginine-nitric oxide (NO) pathway has central, rather than peripheral, mechanisms in blood pressure regulation. Arterial blood pressure and heart rate responses to acute inhibition of the t-arginine-NO pathway were examined in rats anesthetized with thiopental (50 mg/kg, IP). An intracerebroventricular (ICV) cannula was placed in the left lateral ventricle. The right femoral artery was cannulated to measure arterial blood pressure and the vein to serve as an infusion route. $N^G-nitro-L-arginine$ methyl ester (L-NAME) was infused either intracerebroventricularly or intravenously. ICV infusion $(1.25\;{\mu}L/min)$ of L-NAME $(20\;or\;100\;{\mu}g/kg)$ per minute for 60 min) increased the mean arterial pressure and heart rate. Plasma renin concentrations(PRC) were significantly lower in L-NAME-infused group than in the control. L-Arginine $(60\;{\mu}g/min,\;ICV)$ prevented the pressor response to ICV L-NAME. The pressor response was not affected by simultaneous intravenous infusion of saralasin, but was abolished by hexamethonium treatment. Intravenous infusion $(40\;{\mu}L/min,\;10{\sim}100\;{\mu}g/kg\;per\;minute\;for\;60\;min)$ also increased blood pressure, while it decreased heart rate. These results indicate that endogenous L-arginine-NO pathway has separate central and peripheral mechanisms in regulating the cardiovascular function. The central effect may not be mediated via activation of renin-angiotensin system, but via, at least in part, activation of the sympathetic outflow.

Using the methods of stopped-flow and epifluorescence microscopy with entrapped fluorophore, membrane permeability of $NH_3$ and weak acids in liposomes, renal brush border (BBMV) and basolateral membrane vesicles (BLMV), and primary culture cells from renal proximal tubule was measured. Permeability coefficient (cm/sec) of $NH_3$ was $(2.9{\times}10^{-2}$ in phosphatidylcholine liposome $25^{\circ}C)$, $5.9{\times}10^{-2}$ in renal proximal tubule cell $(37^{\circ}C)$, $4.0{\times}10^{-2}\;and\;2.4{\times}10^{-2}$ in BBMV and BLMV $(25^{\circ}C)$, respectively. Formic acid has the highest permeability coefficient among the weak acids tested, which was $4.9{\times}10^{-3}$ in liposome, $5.0{\times}10^{-3}$ in renal proximal tubule cell, $9.1{\times}10^{-3}$ in BBMV and $3.8{\times}10^{-3}$ in BLMV. There was a linear relationship between external concentration of nonionized formic acid and initial rate of flux of formic acid in liposome, and the slope coincided with the value of permeability coefficient of formic acid measured in pH 7.0. These results show that techniques of stopped-flow and epifluorescence microscopy with entrapped fluorophore provide the precise method of measurement of very rapid transport of nonelectrolytes through membranes with the advantages of instantaneous mixing effect, good resolution time and easy manipulation.

The presence of a calcium current $(i_{Ca^{2+}})$ passed via a specific channel was examined in the unfertilized hamster egg using the whole-cell voltage clamp technique. Pure inward current was isolated using a $Ca^{2+}-rich$ pipette solution containing 10 mM TEA. This current was independent of external $Na^+$ and was highly sensitive to the $Ca^{2+}$ concentration in the bathing solution, indicating that the inward current is carried by $Ca^{2+}$. The maximal amplitude was $-4.12{\pm}0.58nA\;(n=12)$ with 10mM $Ca^{2+}$ at -3OmV from a holding potential of -8OmV. This current reached its maximum within 20ms beyond -3OmV and decayed rapidly with an inactivation time constant $({\tau})$ of 15ms. Activation and inactivation of this $i_{Ca^{2+}}$ was steeply dependent on the membrane potential. The $i_{Ca^{2+}}$ began to activate at the lower voltage of -55 mV and reached its peak at -35 mV, being completely inactivated at potentials more positive than -40 mV. These result suggest that $i_{Ca^{2+}}$ in hamster eggs passes through channels with electrical properties similar to low voltage-activated T-type channels. Other results from the present study support this suggestion; First, the inhibitory effect of $Ni^{2+}\;(IC_{50}=13.7\;{\mu}M)$ was more potent than $Cd^{2+}\;(IC_{50}=123\;{\mu}M)$. Second, $Ba^{2+}$ conductance was equal to or below that of $Ca^{2+}$. Third, $i_{Ca^{2+}}$ in hamster eggs was relatively insensitive to nifedipine $(IC_{50}=96.6\;{\mu}M)$, known to be a specific t-type blocker. The physiological role of $i_{Ca^{2+}}$ in the unfertilized hamster eggs remains unclear. Analysis from steady-state inactivation activation curves reveals that only a small amount of this current will pass in the voltage range $(-70{\sim}-30\;mV)$ which partially overlaps with the resting membrane potential. This current has the property that it can be easily activated by a weak depolarization, thus it may trigger a certain kind of a intracellular event following fertilization which may cause oscillations in the membrane potential.

The present study was undertaken to investigate the role of endogenous nitric oxide in renin release under different physiological conditions. In the first series of experiments, renin release was either inhibited by acute volume-expansion (VE) or stimulated by clipping one renal artery in the rat. VE was induced by intravenous infusion of saline (0.9% NaCl) up to 5% of the body weight over 45 min under thiopental (50 mg/kg, IP) anesthesia. VE caused a decrease of plasma renin concentration (PRC). With $N^G-nitro-L-arginine$ methyl ester $(L-NAME,\;5\;{\mu}g/kg\;per\;min)$ superadded to VE, PRC decreased further. The magnitude of increase in plasma atrial natriuretic peptide levels following VE was not affected by the L-NAME. In two-kidney, one clip rats, L-NAME-supplementation resulted in a decrease, and L-arginine-supplementation an increase of PRC. Plasma atrial natriuretic peptide levels were significantly lower in the L-arginine group than in the control. Blood pressure did not differ among the L-NAME, L-arginine, and control groups. In another series of experiments, the renin response to a blockade of NO synthesis was examined using in vitro preparations from isolated renal cortex. L-NAME significantly increased basal renin release, although it was without effect on the isoproterenol-stimulated release. These findings suggest that endogenous nitric oxide significantly contributes to the renin release. Since many factors may affect the renin release in vivo, an interaction between NO and renin under various pathophysiological states is to be further defined.

In this study, it was investigated whether immortalized proximal tubule cells transformed with pRSVT could survive through the numerous passages. Results were as follows: 1. The cells transfected with pRSVT formed rapidly growing, multilayered colonies within 2 weeks in a hormone defined medium. Domes were also observed in some of the cultures. 2. r-glutamyl transpeptidase activity was equivalent to that observed in primary renal proximal tubule cell cultures. 3. Transformed cells with pRSVT form tubules in matrigel following 20 passages. 4. Genomic DNA of transformants was digested with either the restriction enzyme Xba or BamH1. A band of approximately 7.5kb was detected with Xba. Three BamH1 bands were detected at approximately 15 kb, 6.5 kb, and 3 kb.