Introduction

Top Abstract Introduction Methods Results Discussion References

An acute administration of the alpha-2 adrenergic agonist, clonidine, an imidazoline derivative usually used as an antihypertensive drug, produces natriuresis and diuresis in experimental animals and humans (Gehr et al., 1986; Schmitt, 1977). It has been suggested that these renal effects and the antihypertensive action are caused by the activation of centrally located alpha-2 adrenoceptors (Kobinger, 1978; Schmitt and Schmitt, 1970). There is also evidence that central opioid receptors may also be involved in centrally induced renal and cardiovascular effects of clonidine (Farsang and Kunos, 1979; Mastrianni and Ingenito, 1987). However, recent studies showed that the antihypertensive effect of clonidine is mediated through stimulation of another class of receptors that are distinct from alpha-2 adrenoreceptors. Clonidine binds with high affinity to imidazoline binding sites in the rostral ventrolateral medulla oblongata (Michel and Insel, 1989; Molderings et al., 1991).

The diuretic effect of clonidine is mediated by ANF (Pan and Gutkowska, 1988), a potent diuretic, natriuretic and vasorelaxant hormone (De Bold et al., 1981; Ballermann and Brenner, 1985). We have demonstrated that this effect is caused by the activation of central opioid receptors (Pan and Gutkowska, 1988). The medulla oblongata has been suggested as a site of hypotensive and bradycardic action of clonidine (Kobinger, 1978; Laubie and Schmitt, 1977). Although a central effect of the alpha-2 adrenergic agonist, clonidine, is generally accepted, there is also evidence that clonidine has peripheral effects. Significant antinociception was promoted by a clonidine analog, which does not cross the blood-brain barrier (Blandford and Smyth, 1989; Nakamura and Ferreira, 1988). Imidazoline receptors have also been shown to be present in peripheral tissues such as bovine adrenal medulla and rabbit kidney (Wang et al., 1992; Limon et al., 1992). These observations prompted us to investigate whether clonidine exerts its cardiorenal effects through peripheral mechanisms that involve ANF.

Consequently, we used the clonidine structural analog, ST-91, with alpha-2 adrenergic activities, which does not cross the blood-brain barrier (Kobinger and Pichler, 1975). Experiments were undertaken to characterize the effects of ST-91 on water and electrolyte excretion in conscious, normally hydrated rats with use of various doses of ST-91; and also to determine whether the cardiovascular and renal effects of ST-91 are mediated by ANF.

	Methods

Top Abstract Introduction Methods Results Discussion References

Experimental protocol. Experiments were performed on conscious, normally hydrated female Sprague-Dawley rats weighing 200 to 250 g. The animals were housed four per cage at 22°C under 12-h light/dark cycle (room illumination from 6:00 A.M. to 6:00 P.M.). Purina laboratory chow (Ralston-Purina, St.Louis, MO) and water were available ad libitum before the experiment.

Materials. ST-91 hydrochloride (kindly provided by Boehringer Ingelheim Laboratories, Ontario, Canada), yohimbine hydrochloride (Sigma, no. Y3125), naloxone hydrochloride (Dupont Canada NEN, Mississauga, Ontario, no. 1241) and efaroxan (Sigma, no. E3263) were prepared in 0.9% sterile saline immediately before the injection.

Statistical analysis. Data storage, graphical output and statistical analysis assessed by two-way analysis of variance were accomplished with RS1 data analysis software (BBN, Cambridge, MA). Statistical significance was taken as P < .05. All data are reported as means ± S.E.M.

	Results

Top Abstract Introduction Methods Results Discussion References

Intravenous injection of ST-91 to conscious, normally hydrated rats evoked a dose-related increase in urine output during 4 h of treatment. Cumulative urine output over 4 h increased by 7-fold with the dose of 250 µg of ST-91 as compared with values obtained in control animals (1.2 vs. 8.4 ml/4 h, P < .001, n = 18) (fig. 1). However, the most prominent effect was observed during the first hour (fig. 2), in which a 15-fold increase in urine volume was noted with 250 µg of ST-91 (0.2 ± 0.1 vs. 3.0 ± 1.1 ml/h, P < .001, n = 18). With the highest dose of 250 µg, significant diuretic effect was also seen during the second hour (0.3 ± 0.1 vs. 3.0 ± 1.0 ml, P < .001).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of increasing doses of ST-91 on urine output measured during 4 h after drug administration in conscious, normally hydrated rats. (Values are mean of 18 rats/group.)

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of increasing doses of ST-91 on urine output, sodium, potassium and cGMP excretions, during the first hour after drug administration in conscious, normally hydrated rats (n = 18 rats/group). Values are expressed as mean ± S.E.M., *P < .001 vs. saline treatment.

Figure 2 also shows that urinary sodium excretion increased by 12-fold from 28 ± 4 µmol/h in control rats to 401 ± 37 µmol/h (P < .001) with the dose of 100 µg of ST-91 and plateaued with 250 µg (344 ± 49 µmol/h). Similarly, potassium excretion increased during the first hour after ST-91 injection (250 µg) from 10 ± 4 to 165 ± 37 µmol/h (P < .001) (fig. 2).

Urinary cGMP, an index of ANF activity, was measured in experimental and control groups of animals during the first hour after ST-91 administration. Urinary cGMP was augmented in a dose-related manner, from 191 ± 21 pmol/h in control animals, to 1340 ± 322 pmol/h (n = 18, P < .001) during the first hour after ST-91 (100 µg) administration (fig. 2).

These renal effects paralleled the significant 14-fold increase in plasma ANF measured 10 min after administration of 100 µg ST-91 (59 ± 7 vs. 810 ± 28 pg/ml, P < .001, n = 12) (fig. 3). Moreover, compared with rats that received only saline injections, 10 µg ST-91 resulted in elevated plasma ANF levels, and this elevation was sustained during 1-h posttreatment (41 ± 1 vs. 115 ± 15 pg/ml, P < .01, n = 6).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of increasing doses of ST-91 on plasma ANF levels 10 min after drug administration (n = 12 each). Values are expressed as mean ± S.E.M. *P < .02, **P < .001 vs. corresponding saline treatment.

The possible hemodynamic effects of ST-91 such as an increase in blood pressure which could induce ANF release are unlikely. Blood pressure measurement in conscious rats treated with three different doses of ST-91 (1, 2.5 and 10 µg) revealed mild dose-dependent reduction at 10 min after treatment (fig. 4). Blood pressure returned to basal levels at 20 min with the lower doses of ST-91, but remained at subbasal levels (122.4 ± 3.7 vs. 109.6 ± 5.7 and 113.1 ± 4.5 mm Hg) at 20 and 40 min, respectively, when the highest dose (10 µg) was used.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of 1, 2.5 and 10 µg of ST-91 as compared with saline treatment on blood pressure. Values are expressed as mean of four rats in each treatment.

The implication of the peripheral opioid receptors in the action of ST-91 was tested by using naloxone. When the animals were pretreated with the opioid antagonist naloxone at relatively high dose of 0.8 mg/rat, 10 min before ST-91 (10 µg) administration, no alteration in urine output or sodium and potassium excretion was observed (fig. 5), which indicated that the opioid receptors are not involved in the peripheral action. On the other hand, the involvement of alpha-2 adrenergic receptors in the enhanced urinary output and electrolyte excretion in response to ST-91 was determined with use of the alpha-2 adrenergic antagonist yohimbine. Intravenous pretreatment with yohimbine (50 µg) 10 min before ST-91 (10 µg) partially, yet significantly blocked the diuresis, natriuresis and kaliuresis (fig. 5) that were evoked by ST-91 administration. However, efaroxan, an imidazoline receptor antagonist, inhibited the renal responses to ST-91 (10 µg) administration most effectively (fig. 5), which implies that it is mainly the peripheral imidazoline receptors that are involved in the renal responses to ST-91. Furthermore, the ST-91- stimulated excretion of cGMP was inhibited by efaroxan but not by yohimbine or naloxone (fig. 5), suggesting that ANF may be involved in imidazoline but not ₂ adrenergic receptor-mediated actions.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Urine output, sodium, potassium and cGMP excretions during the first hour of treatment with either 50 µg yohimbine, 0.8 mg naloxone or 0.5 mg efaroxan administered before ST-91 (10 µg) injection in conscious, normally hydrated rats (n = 12 each). Values are expressed as mean ± S.E.M. +P < .0001 vs. control, saline-receiving animals. *P < .002, **P < .001 vs. animals receiving ST-91.

Pretreatment with anti-ANF serum (0.4 ml) 10 min before ST-91 (100 µg) administration did not affect sodium excretion but significantly (more than 50% inhibition, P < .05) blocked the ST-91-induced diuresis and natriuresis during the first hour of treatment (fig. 6). The antinatriuretic effect decreased with time but the antidiuretic effect was maintained over 4 hours. Blocking the renal effects of ST-91 by ANF antibody confirms the role of ANF in the actions of ST-91.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Urinary output and sodium excretion induced by ST-91 (100 µg) with or without pretreatment with anti-ANF serum (0.4 ml) 10 min before administration of ST-91. *P < .05 vs. ST-91 alone (n = 6 each).

	Discussion

Top Abstract Introduction Methods Results Discussion References

The present studies provide strong evidence that the renal responses evoked by the peripheral actions of ST-91, a structural analog of clonidine with alpha-2 adrenergic activities, are mainly mediated by the imidazoline receptors and partially by adrenergic receptors through the stimulation of ANF release. These findings were demonstrated by a) the dose-dependent enhancement of diuresis, natriuresis and kaliuresis, as well as plasma ANF and its biological marker cGMP, by peripheral injections of the clonidine analog, ST-91, which does not cross the blood-brain barrier; b) the increased urine output induced by ST-91, which was blocked by anti-ANF; and c) the inhibition of the renal effects of ST-91 by efaroxan which was associated with reduced cGMP excretion. The opioid antagonist, naloxone, showed no effect on diuresis or natriuresis induced by ST-91.

Clonidine, 2-(2,6-dichlorophenylamino)-2 imidazole hydrochloride, is an antihypertensive drug believed to act by inhibiting the sympathetic nervous activity, concomitant with an increase in vagal tone, both effects being attributed to the stimulation of centrally located alpha-2 adrenoceptors (Onesti et al., 1969; Schmitt and Schmîtt, 1970). Further studies accumulated evidence that the antihypertensive action of clonidine is also caused by the involvement of the central opioid receptors (Farsang and Kunos, 1979; Mastrianni and Ingenito, 1987), because the opioid antagonist naloxone attenuates the hypotensive effects of clonidine in spontaneously hypertensive rats (Naranjo et al., 1985). In addition to its function in the neuronal pathway in the brain, clonidine may act on the brainstem (Kunos et al., 1981) and the corticotrophs of the anterior pituitary (Vale et al., 1978) to release -endorphins which may further contribute to the antihypertensive action of clonidine. The hypotensive effect of clonidine is inhibited by intracerebroventricular administration of -endorphin antiserum (Ramirez-Gonzalez et al., 1983; Naranjo et al., 1985).

A general consensus has been reached that the brainstem is the site of cardiovascular action of clonidine, specifically in the nucleus tractus solitarius or ventrolateral medulla. Clonidine promotes analgesia (Paalzow and Paalzow, 1976; Fielding et al., 1978) through both supraspinal (Paalzow, 1974) and spinal levels (Hare and Franz, 1983), and blocks neurophysiological and behavioral symptoms of opioid withdrawal (Aghajanian, 1978; Gold et al., 1978) by central mechanisms. However, analgesia is also promoted by a structural analog of clonidine, ST-91, which does not cross the blood-brain barrier (Bentley et al., 1977). ST-91 is more potent than clonidine in stimulating postjunctional alpha adrenoceptors (Kobinger and Pichler, 1975) and is equipotent with clonidine in stimulating prejunctional inhibitory alpha adrenoceptors (Scriabine et al., 1977). Nakamura and Ferreira (1988) tested ST-91 on hyperalgesia induced by intraplanar injection of prostaglandin E₂ or carrageenin. The antinociceptive effect of ST-91 was dose-dependent, possibly mediated by enkephalin-like substances and indicated peripheral actions.

We and others have previously shown that ANF, whose main source is the cardiac atria, is involved in the cardiorenal effects of clonidine (Baranowska et al., 1987a,b, 1988; Pan and Gutkowska, 1988; Ferrari and Agnoletti, 1989); and that opioids are important stimuli of ANF release, findings confirmed by others (Horky et al., 1985; Gutkowska et al., 1986; Crum and Brown, 1988; Chen et al., 1989; Ogutman et al., 1990). Therefore, we hypothesized that the structural clonidine analog, ST-91, may, by direct effect on the heart or by indirect mechanism via opioids, induce the release of ANF. In fact, the results of the present study show that the diuretic and natriuretic effects of ST-91 were evoked by peripheral mechanisms and that ANF is an element in the cascade of events. However, the opioid receptors are not involved in these effects. It is important to note that the present studies were performed on conscious, normally hydrated animals in which the effect of anesthesia, which may alter the responses to the drugs, was eliminated. Microinjection of clonidine into nucleus tractus solitarius produced pressor responses in conscious animals (Kubo and Misu, 1981) contrary to depressor effect in anesthetized animals (Vlahakos et al., 1985).

The enhanced renal responses to ST-91 were not blocked by naloxone, indicating that peripheral opioids are not a part of the mechanisms of action of the clonidine analog, ST-91. This is compatible with several studies in normotensive humans (Watkins et al., 1980; Pedrinelli et al., 1985) and animals (Farsang et al., 1980; Elghozi et al., 1981; Shropshire and Wendt, 1983) where naloxone does not antagonize the effect of clonidine. Moreover, the lack of naloxone effect observed in our study is in agreement with Vollmar et al. (1987) and Mosqueda-Garcia and Kunos (1988), who suggested that peripherally located opioid receptors are unlikely to be involved in the cardiovascular effects of clonidine or ANF release.

We are proposing a new peripheral mechanism for the diuresis and natriuresis induced by the clonidine analog, ST-91, namely that the renal effects are caused by an enhanced release of ANF that is independent of systemic hemodynamic changes. The mechanisms that mediate the renal actions of ANF may include increased glomerular filtration rate, inhibition of distal sodium reabsorption or medullary washout. The relative importance of these mechanisms is beyond the scope of this study. However, to get some insight into the mechanisms by which ST-91 enhances ANF release, we used efaroxan, an imidazoline receptor antagonist. Indeed, our studies show that both the peripheral alpha-2 adrenoceptors and, more importantly, peripheral imidazoline receptors are activated by ST-91, because the renal effects are mainly inhibited by efaroxan and partly by yohimbine. Studies carried out on isolated atria (Nishimura et al., 1990; Garcia et al., 1986; Gibbs, 1987), atrial cardiocytes (Gibbs, 1987) or in intact animals (Rankin et al., 1987) show that beta adrenoceptors promote the release of ANF. Whether specific imidazoline receptors are found in the heart (Fuder and Schwarz, 1993) and whether the activation of these receptors or the heart alpha-2 adrenoceptors (Fuder and Schwarz, 1993; Starke et al., 1989) could be responsible for the direct release of ANF has to be determined, especially that ANF is synthesized and released from other organs (Gutkowska and Nemer, 1989).

Interestingly, ST-91 induced an important increase in urinary cGMP excretion which supports the role of activated ANF system in the renal responses. The enhanced excretion of cGMP was blocked by efaroxan. Yohimbine tended to decrease urinary cGMP, but naloxone had no effect. Therefore, these findings suggest that it is mainly the imidazoline receptors that are involved in ANF release and subsequent renal responses to ST-91. However, the role of alpha-2 adrenergic receptors can not be ruled out. Molecular cloning showed at least three subtypes of alpha-2 adrenergic receptors in rats, only two of which exhibit a high affinity to yohimbine (Harrison et al., 1991; Uhlen and Wikberg, 1991).

In summary, we propose that ST-91, a clonidine analog with partial alpha-2 adrenergic properties, acts peripherally on the renal system to enhance diuresis and natriuresis. These actions may be mediated by the ST-91-induced release of plasma ANF, through activation of peripheral imidazoline receptors, independently of peripheral opioid receptors.