Int. Med J Vol. 4 No 1 June 2005

NEURAL CONTROL OF RENAL HAEMODYNAMICS IN CARDIAC FAILURE INDUCED SPRAGUE DAWLEY RATS.

S.A. Abbas[a], A.S. Munavvar[b], N.A. Abdullah[c]. and E.J. Johns[d]

ABSTRACT

We have reported earlier the involvement of α-adrenoceptors at the level of renal resistance vessels to regulate the renal blood flow in cardiac failure induced SD rats (Atif et al 2001). The present study was designed to determine the functional α-adreneroceptor subtype in the renal vasculature at the level of the arterioles of the SD rats with cardiac failure. The cardiac failure was induced by the combined treatment of caffeine (20mg/kg) and isoproterenol (5mg/kg) for seven days. On the day eight the rats were used for the acute study. The animal was anaesthetised with pentobarbitone sodium (60mg/kg IP). After tracheotomy the left jugular vein and carotid artery were cannulated to allow the continuous infusion of anaesthesia and to measure the arterial blood pressure respectively. The left kidney was exposed by moving carefully the abdominal contents to right side following a midline abdominal incision. The renal artery was cleared and an electromagnetic flow probe was placed on it to determine the renal blood flow (RBF). The left iliac artery was cannulated such that the beveled tip of cannula faced the renal artery for the infusion of saline and all drugs close renal arterially. The renal nerves were identified, isolated and placed on bipolar electrodes for electrical stimulations. Upon completion of surgery 2ml of saline was injected intravenously as primer. The reduction in RBF to electrical nerve stimulation (at 1,2,4,6,8 and 10 Hz at 15 V and 2ms), bolus doses of noradrenaline(NA) (25, 50, 100 and 200 ng), phenylephrine(PE) (0.25,0.5,1.0 and 2.0 µg) and methoxamine(ME) (1,2,3 and 4 µg) were determined before and after bolus doses of amlodipine(AMP) (200 and 400 µg /kg plus 50 and 100 µg /kg/h), 5-methylurapidil(5MEU) (5 and 10   µg /kg plus 0.125 and 0.25 µg /kg/h), chloroethylclonidine(CEC) (5 and 10 µg /kg plus 0.125 and 0.25 µg /kg/h) and BMY7378 (100 and 200 µg /kg plus 25 and 50 µg /kg/h). Data, means ± s.e.m were compared with 2 way ANOVA followed by Bonferroni post hoc with the significance level of 5%. The results obtained indicated that the renal vasoconstrictor responses in this model were attenuated by amlodipine, 5-methylurapidil and BMY7378 but not by chloroethylclonidine. These findings showed that in cardiac failure induced SD rats the greater part of adrenergic vasoconstriction in kidney of these animals was mediated by α 1A and
α 1D - adrenoceptors with a minor involvement of α 1B - adrenoceptors.

INTRODUCTION

Adrenoceptors mediate the central and peripheral actions of noradrenaline and adrenaline. Adrenoceptors are found in most of the peripheral tissues and neurons within the central nervous system. These adrenoceptors mediate a variety of functions such as blood pressure and myocardial contractile rate and force.

In the kidney the a- adrenoceptors modulate renal blood flow, electrolyte balance and metabolism. The a1- adrenoceptors control blood flow and increase sodium reabsorption and gluconeogenesis. On the other hand a2- adrenoceptors inhibit adenylate cyclase, and  their density varies among species as do the molecular characteristics (Summers., 1984). In the kidney the existence of multiple adrenoceptors subtypes have been reported (Minneman et al., 1988; Han et al., 1990; and Feng et al., 1991). The proportion of a1A and a1B- adrenoceptors is almost equal in the cortex and outer stripes of medulla, but with the a1B- subtype predominating in the inner stripe of the outer medulla, whilst at the proximal tubules they are expressed at approximately equal levels (Feng et al., 1991). Three a1- adrenoceptor subtype mRNAs were recognized in human renal cortex and detected particularly in the smooth muscles of the arteries (Kurooka et al., 1999). There were more a1A- adrenoceptor subtypes in human renal cortex than the other subtypes. Expression of the three a1- adrenoceptor subtypes mRNAs were confirmed in the arteries of the renal cortex, but among the three subtypes the a1B was less apparent by in situ hybridization. Intense a1-mRNA staining was apparent especially in the smooth muscles of the arterial walls of the kidney. In both proximal and distal renal tubules, each of the a1-mRNAs were less marked in cytoplasm than in the arteries. In the glomeruli, weak staining was detected in the endothelium but there was no obvious staining in the vein. Reverse transcryptasepolimerase chain reaction showed all the three subtypes of  a1- adrenoceptor in the rat renal cortex.  Similarly, Moriyama et al., (2000) showed that the predominant a1- adrenoceptor subtype mRNA in human renal cortex was a1A. The role of a1- adrenoceptors in mediating the rat renal vasoconstriction has been well documented (Han and Minneman, 1990 and Sattar and Johns, 1996) and it has been revealed that the adrenergically induced vasoconstriction was primarily mediated by the a1A- adrenoceptors in normotensive (Sattar and Johns, 1994a; Blue et al., 1995), SHR, SPSHR and 2 kidney one clip Goldblatt and DOCA-salt hypertensive rats (Sattar and Johns, 1994a and b).  These findings are sported by Zhu et al., (1997) in which they found that the a1A- adrenoceptor is the major subtype in renal resistance arterioles. Other studies have also shown that the sympathetic nerve stimulation of the perfused kidney provokes vasoconstriction which is extremely sensitive to low concentration of a1A- adrenoceptor antagonist but not CEC (Khong et al., 1994). In vessels such as renal artery, the a1- adrenoceptor may be located at the neurovascular synapses where they receive direct sympathetic nerve innervations (Williams and Clarke, 1995).

However, there is little or no information available regarding the contribution of a1- adrenoceptor subtypes in mediating renal vasoconstriction in cardiac failure and its combination with diabetes.

This study is one the first to examine the contribution of a1- adrenoceptor subtype(s) in mediating the adrenergically induced renal vasoconstrictor responses in cardiac failure  induced Sprague Dawley and spontaneously hypertensive rats.

METHODOLOGY

Male Sprague Dawley rats (300-325g) were used in this study. Cardiac failure was induced by the combined treatment of caffeine (40mg/kg) and isoprenaline (5mg/kg) for seven days. On day eight the rats were used for acute study. The animal was anaesthetised with pentobarbitone sodium (60mg/kg IP). The right carotid artery was cannulated for blood pressure measurements (Statham Grass model 79E polygraph). The left jugular vein was cannulated to allow infusion of saline and maintenance dose of anaesthetic (12.5mg/kg/h) which was given at the rate of 6 ml/h throughout the experiment.

The left kidney was exposed by an abdominal mid-line incision. The left iliac artery was cannulated with PP50, with a beveled tip which was advanced into the abdominal aorta, such that it lay at the level of the renal artery to enable the infusion of saline and administration of all drugs close renal arterially. The renal artery was cleared and an electromagnetic flow probe (EP 100 series) was placed on it. The probe was connected to a Square-Wave Electromagnetic Flow meter (Model FM 501 King, NC, Carolina Medical Electronic Inc) for renal blood flow measurements. The left renal nerves, passing from the coeliac and aortico- renal ganglia, were identified, cleaned of peritoneal lining, dissected for a short length to enable them to be placed on a bipolar silver stimulating electrode and sectioned.  The functionality of the renal nerves was established before and after sectioning by stimulating the bundle for 15-30 seconds and observing that a blanching of the kidney occurred.

Upon completion of surgery, 2ml of saline was given intravenously as a primer and an infusion of saline containing sodium pentobarbitone 12.5mg/kg/h was started at a rate of 6ml/h via the close renal arterial cannula.  The animals were given one hour to stabilize before the commencement of experiment.

EXPERIMENTAL PROTOCOLS

Four groups of rats were studied in this experiment and renal vasoconstrictor responses were determined by:

1. Electrical renal nerve stimulations

2. Different doses of agonists (NA, PE and ME)

The reduction in RBF to electrical nerve stimulation (at 1, 2, 4, 6, 8 and 10 Hz at 15 V and 2ms), bolus doses of noradrenaline (25, 50, 100 and 200 ng), phenylephrine (0.25, 0.5, 1.0 and 2.0 mg) and methoxamine (1, 2, 3 and 4 mg) were determined before and after bolus doses of antagonists i.e amlodipine (200 and 400 mg/kg plus 50 and 100 mg/kg/h), 5 methylurapidil (5 and 10 mg/kg plus 1.25 and 2.5 mg/kg/h), chloroethylclonidine (5 and 10 mg/kg plus 1.25 and 2.5 mg/kg/h) and BMY7378 (100 and 200 mg/kg plus 25 and 50 mg/kg/h).

Renal nerve stimulations

The renal nerves were stimulated at a number of different frequencies, 1, 2, 4, 6, 8 and 10 Hz, at 0.2 ms duration and 15 V for periods of 20 seconds and in a sequence of ascending followed by descending frequencies such that two responses at each frequency was obtained.

 

Different doses of agonists (NA, PE and ME)

Bolus doses of NA, 25, 50, 100, and 200 ng, PE,0.25, 0.5, 1.0 and 2.0 µg and ME, 1, 2, 3, and 4 µg were given close renal arterially in increasing and decreasing doses such that two responses per dose of the agonist were obtained.  The determination of control renal vasoconstrictor responses to renal nerve stimulation, NA, PE and ME were carried out 15 minutes after the administration of the bolus dose of the vehicle.  When the control responses had been recorded, the first dose of antagonist was given immediately followed by the respective infusion and 15 minutes later the sequence of nerve stimulation and agonist administration was repeated.  The second dose of the antagonist was then given and the supporting infusion started immediately and after 15 minutes the sequence of renal vasoconstrictor responses induced by renal nerve stimulation and the agonists determined.  A renal vasoconstrictor response was taken as the peak reduction in the renal blood flow to either direct renal nerve stimulation or the agonist and the average of the two values obtained were used for the calculation. The renal blood flow was allowed to return to normal (baseline) level before commencing with subsequent renal nerve stimulation or of agonist administration.  The renal vasoconstrictor responses obtained after bolus injection of vehicle served as controls for each experimental group. The doses of agonists and antagonists were adopted from the previous and our own preliminary studies (Sattar and Johns, 1994a).

 

Mean Arterial Pressure (MAP)

The mean arterial pressure was monitored continuously throughout the experiment, but only that at the beginning of each part of vasoconstrictor response was taken as the base line pressure.

Preparation of Drugs

CEC, AMP and BMY7378 were dissolved in normal saline, whereas 5MeU was dissolved in 10 mmol lactic acid in normal saline (Sattar and Johns, 1996). All the agonists and antagonists were prepared fresh before starting the experiment.

STATISTICAL ANALYSIS

The renal vasoconstrictor responses to renal nerve stimulation, noradrenaline, phenylephrine and methoxamine were taken as the average of the values obtained during the ascending and descending frequencies and doses in the absence and presence of antagonist. The overall mean response was taken as the average value of renal vasoconstrictor responses to renal nerve stimulation and the agonists at all frequencies and doses respectively. All data was expressed as mean %change ± s.e.m. Statistical analysis of data was performed by a 2-way analysis of variance followed by Bonfferoni’s post hoc test. The differences between the means were considered significant at the 5% level. Statistical analysis was performed using the statistical package, SUPERANOVA.

RESULTS

RENAL VASOCONSTRICTOR RESPONSES:

RNS produced frequency-dependent (P<0.001) decrease in renal blood flow in SD rats. In the low dose of AMP and BMY7378 there was no significant (P>0.05) change on the renal blood flow from the lowest to highest (1-10Hz) applied frequencies of RNS as compared to control in SD rats. Whereas the high doses of AMP and BMY7378 caused a significant (P<0.05) change on the renal blood flow from the lowest to highest (1-10Hz) applied frequencies of RNS as compared to control. However, high and low doses of 5MeU and CEC resulted in significant (P<0.05) change in the renal blood flow from the lowest to highest (1-10Hz) applied frequencies of RNS as compared to control in SD rats. (Fig. 3.5, 3.6, 3.7 and 3.8)

 

 

 Noradrenaline (NA)

In general, NA induced vasoconstrictor responses were dose dependent (P<0.001) in the SD rats. 

In general, NA induced vasoconstrictor responses were dose dependent (P<0.001) in the SD rats. BMY7378 and AMP in both low and high doses caused a significant change (P<0.05) on the renal blood flow from the lowest to highest doses of NA as compared to control in SD rats. Whereas in case of 5MeU low dose does not cause any change in RBF from the lowest to highest doses of NA as compared to control in SD rats but high dose caused significant change (P<0.05) on the renal blood flow from the lowest to highest doses of NA as compared to control in SD rats. In CEC treated group of rats low dose caused significant change (P<0.05) on the renal blood flow from the lowest to highest doses of NA as compared to control in SD rats. Whereas high dose does not cause any change in RBF from the lowest to highest doses of NA as compared to control in SD rats. (Fig. 3.9, 3.10, 3.11 and 3.12).

 

Phenylephrine (PE)

PE  induced renal vasoconstrictor responses were dose dependent in all groups of SD rats (P<0.001).

PE  induced renal vasoconstrictor responses were dose dependent in all groups of SD rats (P<0.001). AMP, BMY7378, CEC and 5MeU in both low and high doses caused a significant change (P<0.05) in the renal blood flow from the lowest to highest doses of PE as compared to control in SD rats. (Fig. 3.13, 3.14, 3.15 and 3.16).

 

 

Methoxamine (ME)

ME  induced renal vasoconstrictor responses were dose dependent in all groups of SD rats (P<0.001).

ME  induced renal vasoconstrictor responses were dose dependent in all groups of SD rats (P<0.001). AMP, 5MeU and BMY7378, in both low and high doses caused a significant change (P<0.05) in the renal blood flow from the lowest to highest doses of ME as compared to control in SD rats. However, CEC in both low and high doses did not cause a significant change (P>0.05) in the renal blood flow from the lowest to highest doses of ME as compared to control in SD rats. (Fig. 3.17, 3.18, 3.19 and 3.20)

 

DISCUSSION

It has been reported earlier that the renal artery constriction was regulated primarily by a1A- adrenoceptor subtypes. This is based on its resistance to be alkylated by CEC (Piascik et al., 1994) and weakly antagonized by BMY7378 (Piacik et al., 1995). Sattar and Johns (1994a and b) reported that at the level of renal resistance vessels, the constriction was mediated by 5MeU sensitive a1- adrenoceptors which are the a1A- adrenoceptors and later Zhu et al., (1997) supported that a1A- adrenoceptor is the major subtype in renal resistance arterioles.

In the heart failure, desensitization of b- adrenoceptors is related to a lower adrenergic responsiveness in heart. However, little is known about the a- adrenoceptors in the renal vasculature under this condition. Recent studies have shown that a1D- adrenoceptor function is maintained during congestive heart failure after myocardial infarction in rats and a1D- adrenoceptors are the main receptors involved in aorta and carotid arteries, irrespective of congestive heart failure (Martinez and Dunbar, 1999). It has been indicated that the mRNAs for a1D- adrenoceptor subtype exist in the renal artery (Piascik et al., 1994 and 1995; Guarino et al., 1996). This adrenoceptor subtype may play a significant role in the regulation of renal vasoconstriction (Hormetz et al., 1999). Besides playing a role in renal vasoconstriction, a1D- adrenoceptors are also involved in the pathogenesis and maintenance of elevated blood pressure                                       (Villalobos-Mollina et al., 1999).

The constrictor responses of vascular smooth muscles to vasoactive agents in chronic diabetic animals have been widely studied and it was shown that vascular responses to a- adrenoceptor agonists were unchanged, increased or decreased. The variability of the results of the vasoconstrictor responses could probably be due to the differences in procedures, strains, gender and age of rats used, and or duration of diabetes                      (Garcia et al.,1999).

It is clear from the discussion that a1- adrenoceptors play a major role in the regulation of the renal haemodynamics and hence, the present research focused on the  a1- adrenoceptor subtypes involved in the adrenergically induced vasoconstrictions in heart failure induced SD rats.

Electrical stimulations of the renal nerves at the increasing frequencies led to the graded reductions in renal blood flow. Previous investigators have also shown that in the normotensive rats, these responses are mediated by a1- adrenoceptors. It is also demonstrated earlier that in 2K1C and DOCA- salt hypertensive group of rats, the calcium channel blockers attenuated the magnitude of the renal vasoconstrictor responses to nerve stimulation which became larger as the dose of AMP was raised (Sattar M.A and Johns E.J, 1994a). In the present study, in cardiac failure induced SD rats, the high dose of AMP, caused a significant blockage of vasoconstrictor responses induced by renal nerve stimulation.

To further strengthen this view, 5 MeU a specific a1A- adrenoceptor antagonist, was used to block the effect of renal nerve stimulation induced vasoconstriction. The results showed that in cardiac failure induced SD rats the vasoconstrictor effects by RNS were suppressed in a dose related fashion as in case of AMP. These findings further suggested that a­1A- adrenoceptor subtype were involved in the neurally induced vasoconstriction.

In the presence of BMY7378 a a­1D- adrenoceptor antagonist, it was observed that in SD rats only the high dose blocked the vasoconstrictions.

These observations indicated that the renal vasoconstrictions in SD rats with cardiac failure were not only mediated by a­1A- adrenoceptors as described by previous investigators (Blue et al.,  Sattar and Johns, 1994a and b and Blue et al., 1995) but also by a­1D- adrenoceptors based on their sensitivity to BMY 7378.

CEC an a­1B- adrenoceptor antagonist (Han et al., 1987,  Piascik et al., 1995) that acts by alkylating a­1B- adrenoceptor and is an important tool in the sub classification of the a­1- adrenoceptors. In the present study high as well as low doses of CEC caused a significant reduction in the neurally induced vasoconstrictions of SD rats with cardiac failure. This could have been attributed to the blockade of the unsaturated pre-synaptic a- adrenoceptors. This can be explained by the possibility of CEC, being and analogue of clonidine, to act pre-synaptically and that these receptors could be of the a­1B- adrenoceptor subtype. There is a possibility that occupation of a­1B- adrenoceptors lead to an alteration in the properties of a­1A- adrenoceptors such that the normal agonist and antagonist interaction cannot occur. In this situation, the blockade of a­B- adrenoceptors would enhance the sensitivity of the remaining a­1- adrenoceptors such that a potentiation of the renal vasoconstrictor responses were seen. Another possibility is the activation of spare receptors which could have occurred with the blockade of a­1B- adrenoceptors. A similar situation was reported by Piascik et al., (1994) in which phenoxybenzamine incubated alone with the aortic rings completely abolish the response to phenylephrine, whereas following treatment with either SZL49, an a­1A- adrenoceptors antagonist, or CEC, a putative a­1B- adrenoceptor blocking agent, the actions of phenoxybenzamine, which can antagonize both the receptor subtypes was abolished.

Administration of noradrenaline caused dose dependent reductions in renal blood flow. The results showed that in cardiac failure induced SD rats.

AMP, BMY7378, and 5 MeU caused a significant attenuation of the vasoconstrictor effect, by noradrenaline where as CEC a specific a­1B- adrenoceptor antagonist in high and low dose did not exhibit any significant effects on the magnitude of noradrenaline induced vasoconstrictor responses.

According to Moriyama et al., (2000), the a­1A/L- adrenoceptors primarily mediate the vasoconstrictor responses to noradrenaline, although other a­1A- adrenoceptor subtypes could also contribute in the mediation of the secondary contractile response to noradrenaline in the renal artery. RNase protection assay showed that the mean amount of a­1A mRNA was much greater than that of a­1B or a­1D mRNAs in both the main and branch renal arteries. Furthermore, in situ hybridization showed that all a­1- subtype mRNAs were localized in the smooth muscle cells of the tunica media of artery, and the distribution pattern of these three mRNAs in the main artery was the same as in the branch artery. However, the intensity of signals for a­1D and a­1B antisense RNAs probes was lower than that for the a­1A antisense RNA probe. Furthermore, CEC failed to inactivate the noradrenaline-induced contraction, and prazosin showed a relatively low affinity (Moriyama et al., 2000). These findings indicated that the a­1B- adrenoceptors are not involved in mediating renal vasoconstriction.

Administration of phenylephrine which activates both a­1A and a­1B- adrenoceptors, caused dose dependent reductions in renal blood flow in cardiac failure induced SD rats. The renal vasoconstrictor responses to phenylephrine were significantly reduced by AMP, BMY7378 and 5MeU in cardiac failure induced rats but not by CEC. The results obtained  are supported by the previous investigations (Sattar and Johns, 1996) and strengthen the view that the phenylephrine evoked renal vasoconstrictions were mediated by the subtype of a­1- adrenoceptors that were dependent on extracellular calcium influx, that is, the a­1A- adrenoceptors. To support this contention, studies using 5 MeU were utilized. 5MeU in the high and low doses significantly depressed the renal vasoconstrictor responses to exogenous phenylephrine. As phenylephrine is capable of exerting its action on a­1- adrenoceptors and an antagonism of the a­1A- adrenoceptors by 5MeU led to an attenuation of these vasoconstrictor responses, it could be suggested that these responses are mediated by the a­1A- adrenoceptors. On the other hand, CEC did not exhibit any significant effects on the magnitude of the phenylephrine induced renal vasoconstrictor responses. This it can be argued, that the failure of CEC to manifest any meaningful changes would suggest a minor role of the a­1B- adrenoceptors in mediating the phenylephrine induced vasoconstrictor responses. These studies combined provides further support that in cardiac failure SD rats, a­1A- adrenoceptors are functionally important. In cardiac failure and diabetes induced SD rats, CEC also reduced the vasoconstrictor effect induced by phenylephrine. Similar findings  were obtained when AMP, BMY7378 and 5MeU was utilized. The close renal arterial administration of methoxamine, a putative a­1A- adrenoceptor antagonist (Tsujimoto et al., 1989) similarly resulted in dose dependent reductions in renal blood flow in cardiac failure induced SD rats. AMP, BMY7378 and 5 MeU (high doses) administration resulted in suppression of methoxamine induced renal vasoconstrictions in all groups. However, administration of CEC did not show a significant reduction in methoxamine induced renal vasoconstriction in cardiac failure SD rats. This observation further strengthen the view that a­1A- adrenoceptors are involved in renal vasculature of cardiac failure induced SD rats.

These findings supported that differences in a­1- adrenoceptor populations and distribution in blood vessels are dependent on the pathological state (LeTran and Forster, 1997). The findings from this study suggest that the a­1A and a­1D- adrenoceptor mediate the adrenergically induced renal vasoconstrictor responses in cardiac failure SD rats.

 

CONCLUSIONS

These studies collectively provide further support that in cardiac failure induced SD rats a­1A- adrenoceptors are functionally important. The pathological conditions cardiac failure can cause the down regulation of a­1A- adrenoceptors. The renal vasoconstrictions in SD rats with cardiac failure were not only mediated by a­1A- adrenoceptors but also by a­1D- adrenoceptors based on their sensitivity to BMY 7378.

a­1B-adrenoceptors are not involved in mediating renal vasoconstrictions in these animal models.

REFERENCES

  1. Blue, D. R. Jr., Bonchaus, D. W., Ford, A. P. D. W., Pfister, J. R., Syarif, N. A., Shiekh, I. A., Vimont, R. L., Williams, T. J. and Clarke, D. E. (1995). Fuctional evidence equating the pharmacologically-defined a1A and cloned a1A- adrenoceptor: studies in isolated perfused kidney of rat, Br. J. Pharmacol., 115, 283-294.

  2. Feng, F., Pettinger, W. A., Abel, P.W., Jeffries, W. B. (1991). Regional distribution of        a1- adrenoceptor subtypes in rat kidney. J. Pharmacol. Exp. Ther. 258(1), 263-8

  3. Garcia, V. M. C., Ochoa, J. E. and Elias, M. M. (1999). Effect of early stage of experimental   Diabetes on vascular functions in isolated perfused kidneys. J. Auton. Pharmacol., B, 97-103.

  4. Guarino, R. D., Perez, D. M. and Piascik, M. T. (1996). Recent advances in the molecular pharmacology of the a1- adrenergic Receptors. Cell Signal., 8(5), 323-33.

  5. Han, C., Able, P. W. and Minneman, K. P. (1987). Heterogeneity of a1- adrenergic receptors revealed by chloroethylclonidine. J. Pharmacol. Exp. Ther., 32, 505-510.

  6. Han, C., Li, J. and Minneman, K. P. (1990). Subtypes of a1- adrenoceptors in rat blood vessels. Eur. J. Pharmacol., 190, 97-104.

  7. Hormetz, S. L., Edelmann, S. E., McCune, D. F., Olges, J. R., Hadley, R. W., Perez, D. M. and Piascik, M. T. (1999). Statement of multiple a1- adrenoceptors on vascular smooth muscle: Correlation with the regulation of contraction. J. Pharmacol. Exp. Ther., 290 (1), 452-463.

  8. Khong, J. Q., Miller, A. M. and Flemming, W. W. (1994). Functional distribution and role of    a1- adrenoceptor subtypes in the messentric vasculature of the rat. J. Pharmacol. Exp.Ther., 268, 1153-59.

  9. Kurooka, Y., Moriyama, N., Nasu, K., Kameyama, S., Fukasawa, R., Yano, J. and Kawabe, (1999). Distribution of a1- adrenoceptor subtype mRNAs in human renal cortex. BJU Int., 83(3), 299-304.

  10. Le Tran Y., and Forster, C. (1997). Chlorethylclonidine and a- adrenoceptor subtypes mediate constriction of arterioles and venules. Am. J. Physiol., 270 , H711-H722.

  11. Martinez-Nieves, B. and Dunbar, J. C. (1999). Vascular dilatatory responses to sodium nitroprusside (SNP) and a-adrenergic antagonism in female and male normal and diabetic rats. Proc. Soc. Exp. Biol. Med., 222(1), 90-8.

  12. Minneman, K. P., Han, C. and Abel, P. W. (1988). Comparizon of a- adrenergic receptor subtypes distinguished by chlorethylclonidine and WB4101. J. Pharmacol. Exp. Ther., 33, 509-514.

  13. Moriyama, N., Kurooka, Y., Nasu, K., Akiyama, K., Takeuchi, T., Nishimatsu, H., Murata, S. Murayama, T., Tsujimoto, G. and Kawabe, K. (2000). Distribution of a1-adrenoceptor subtype mRNA and identification of subtype responsible for             renovascular contraction in human renal artery. Life Sci., 66(10), 915-26.

  14. Piascik, M. T., Guarino, R. F., Smith, M. S., Soltis, E. E., and Perez, D. M. (1994). Identification of mRNA for the novel a1D- adrenoceptor and the two other a1- adrenoceptors in the vascular smooth muscle. Mol. Pharmacol., 46, 30-40.

  15. Piascik, M. T., Guarino, R. F., Smith, M. S., Soltis, E. E., Saussy, D. L., and Perez, D. M. (1995). The specific contribution of the novel a1D- adrenoceptor to the contraction of vascular smooth muscle. J. Pharmacol. Exp. Ther., 275, 1583-1589.

  16. Sattar, M. A. and Johns, E. J. (1994a). Evidence for an a1-adrenocepter subtype Mediating adrenergic vasoconstriction in Wistar normotensive and SPSHR kidney. J. Cardiovasc. Pharmacol., 23, 232-239.

  17. Sattar, M. A. and Johns, E. J. (1994b). a1- adrenoceptor subtype mediating adre-nergic vasoconstriction in kidney of 2K1C and DOCA-Salt hypertensive rats. J. Cardiovasc. Pharmacol., 24, 420-428.

  18. Sattar, M. A. and Johns, E. J. (1996). a1-adrenocepter subtypes involved in mediating adrenergically induced antinatriuresis in two kidney, one-clip Goldblatt and DOCA-salt hypertensive rats. J. Pharmacol. Exp. Ther., 277, 245-252.

  19. Summers, R. J. (1984). Renal a-adrenoceptors. Fed. Proc., 43, 2917-2922.

  20. Tsujimoto, G., Tsujimoto, A., Suzuki, E. and Hashomoto, K. (1989). Glycogen phosphorylase activation by two different a1-adrenergic receptor subtypes: Methoxamine selectively stimulates a putative a1- adrenergic receptor subtype (a1A) that couples with Ca++       influx. J. Pharmacol. Exp. Ther., 36, 166-176.

  21. Villalobos-Molina, R., Lopez-Guerrero, J. J and Ibarra M. (1999). Functional evidence of a1D- adrenoceptors in vasculature of young and adults spontaneously hypertensive rats. Br. J. Pharmacol., 126(7), 1534-1536.

  22. Williams, T. J., and Clarke, D. E., (1995). Characterization of a1- adrenoceptors mediating vasoconstriction to noradrenaline and nerve stimulation in the isolated perfused mesenteric of rat. Br. J. Pharmacol., 114: 531-536. 

  23. Zhu, W., Zhang, Y. and Han, C. (1997). Characterization of subtype a1- adrenoceptor mediating vasoconstriction in perfused rat hind limb. Eur. J. Pharmacol., 329, 55-61.

[a] Faculty of Pharmacy, International Islamic University Malaysia, Kuantan.

[b] School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia

[c] Department of Pharmacology, Faculty of Medicine University of Malaya Kuala Lumpur

[d] Department of Physiology, University College Cork, College Road Cork, Ireland..