Ismail A. Dreshaj

Selective antihypertensive action of moxonidine is mediated mainly by I1-imidazoline receptors in the rostral ventrolateral medulla

The rostral ventrolateral medulla (RVLM) is the primary region maintaining vasomotor tone, and a site of action for central antihypertensive agents. In vitro [125I]p-iodoclonidine binding studies showed that moxonidine was selective for I1-imidazoline over alpha 2-adrenergic receptors in the RVLM. We identified efaroxan and SK&F 86466 as selective I1- and alpha 2-antagonists, respectively. We tested moxonidines action within the RVLM of spontaneously hypertensive rats (SHRs) on I1-imidazoline or alpha 2-adrenergic receptors, and determined whether the RVLM mediates the action of systemic moxonidine. SHRs were anesthetized, paralyzed, and ventilated and the RVLM was localized by testing for a pressor response to 2 nmol glutamate. To test whether I1 or alpha 2 mediates hypotensive effects of moxonidine, the I1/alpha 2 antagonist efaroxan (4 nmol) or the alpha 2-blocker SK&F 86466 (10 nmol) was administered 15 min before 4 nmol moxonidine. Efaroxan elevated blood pressure and abolished the action of moxonidine, whereas alpha 2-blockade with SK&F 86466 slightly lowered blood pressure and only partially attenuated moxonidines effect. The depressor effect of intravenous moxonidine (40 micrograms/kg) was reversed within 10 min by microinjection of 10 nmol efaroxan into the RVLM. Prior bilateral microinjections of efaroxan (10 nmol in 80 nl/site) into the RVLM prevented the hypotensive action of moxonidine given i.v. (40 micrograms/kg). Pharmacokinetic studies showed that at the peak vasodepressor response (8 min post-injection), [3H]moxonidine spread less than 1 mm from the injection site. Moxonidine is a centrally acting antihypertensive with a selective action on I1-imidazoline receptors in RVLM.

Role of the medullary raphe nuclei in the respiratory response to CO2

We characterized the role of neurons within the midline of the medulla oblongata on phrenic and hypoglossal nerve responses to hypercapnia during early-development. Studies were performed on decorticate or anesthetized; vagotomized and mechanically ventilated 14-20 day old piglets. Reversible withdrawal of midline neuronal activity was induced by microinjections of lidocaine (2%, 300 nl; n = 10) and lesioning was caused by microinjections of the neurotoxic agent, ibotenic acid (n = 12), at the same sites. At any given end-tidal CO2, peak phrenic and hypoglossal activities after lidocaine were significantly lower than in the control period (P < 0.01). Similarly, 1-2 h after injections of ibotenic acid, both phrenic and hypoglossal nerve responses to CO2 were significantly lower than in the control period (P < 0.01). The results indicate for the first time that the medullary midline neurons are required for full expression of ventilatory responses to hypercapnia and raise the possibility that dysfunction of these nuclei may contribute to respiratory instability during early postnatal life.

Nitric oxide and ventilatory response to hypoxia

It is believed that hypoxia results in the release of neurotransmitters in the central nervous system, which can excite or inhibit breathing. Recent evidence indicates that nitric oxide (NO) is a physiological messenger molecule that may serve as a neurotransmitter in the CNS. In this study we examined (1) the localization of nitric oxide synthase (NOS) within the nucleus tractus solitarius, and (2) the role of the NO-cGMP pathway in the respiratory response to oxygen deprivation. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry was used to determine the distribution of neurons that express NOS, an enzyme involved in NO formation. The NOS inhibitor N omega-nitro-L-arginine was used as tool to assess the NOS activity in the medulla, and to define the role of NO in the respiratory response to acute oxygen deprivation. In the rat and the cat brainstem, histochemical studies showed the presence of NADPH-diaphorase reactive neurons within subnuclei of the nucleus tractus solitarius which receive peripheral chemoreceptor inputs. Chronic pretreatment of rats with N omega-nitro-L-arginine (75 mg/kg, ip, twice daily for 7 days) caused a significant decrease in cGMP, and attenuated the ventilatory response to hypoxia. In anesthetized, paralyzed, vagotomized and artificially ventilated cats with intact carotid sinus nerves (n = 8), administration of N omega-nitro-L-arginine (30-100 mg/kg) attenuated the response to hypoxia, and caused the hypoxia induced roll-off of phrenic nerve activity to occur significantly earlier than when NOS activity was not inhibited. In sinoaortic denervated cats (n=9) blockage of NOS potentiated the decline of the phrenic nerve output. The data suggest that oxygen deprivation leads to activation of NO-cGMP pathway in the central nervous system, which contributes to the induction and maintenance of hypoxia-induced increase in respiratory output. In addition, these findings indicate that NO may inhibit inhibitory synaptic transmission that is triggered by CNS hypoxia, and this is not directly related to peripheral chemoreceptor inputs.

A novel mechanism of action for hypertension control: Moxonidine as a selective I1-imidazoline agonist

SummarySympathoadrenal inhibition by a direct action within the central nervous system is an advantageous route to blood pressure control. Stimulation of brain α2-adrenergic receptors is one mechanism for sympathoadrenal suppression, but comes at the cost of nonspecific depression of CNS function, including sedation and decreased salivary flow. Evidence is accumulating for a second pathway for pharmacological control of sympathoadrenal outflow, mediated by a novel receptor specific for imidazolines. First-generation central antihypertensive agents, which are imidazolines such as clonidine, act primarily to stimulate these I1-imidazoline receptors in the rostral ventrolateral medulla oblongata (RVLM) to lower blood pressure, but have sufficient agonism at α2-adrenergic receptors to produce side effects. Second-generation centrally acting antihypertensive agents, such as moxonidine and rilmenidine, are selective for I1 relative to α2 receptors. The reduced α2 potency of these agents correlates with reduced severity of side effects. In this study we further established the selectivity of moxonidine for I1-imidazoline sites by characterizing the direct interaction of [3H]moxonidine with these receptors in the RVLM and in adrenomedullary chromaffin cells. [3H]Moxonidine preferentially labeled I1-imidazoline sites relative to α2-adrenergic sites, only a small portion of which were labeled in the RVLM. [3H]Moxonidine binding to I1-imidazoline sites was modulated by guanine nucleotides, implying that I1-imidazoline sites may be membrane receptors coupled to guanine nucleotide binding regulatory proteins (G proteins). Receptor autoradiography with [125I]p-iodoclonidine confirmed the presence of I1-imidazoline sites in the RVLM and other areas of the brainstem reticular formation. In contrast, α2-adrenergic sites were mainly localized to the nucleus of the solitary tract. Moxonidine selectively displaced [125I]p-iodoclonidine binding from reticular areas, including the RVLM. In vivo studies in SHR rats confirmed the ability of moxonidine to normalize hypertension by an action within the RVLM and confirmed the correspondence of I1 binding affinity and antihypertensive efficacy. We also discuss prior literature on the cardiovascular pharmacology of imidazolines, reinterpreting previous studies that only considered alpha-adrenergic mechanisms.

Effect of Exogenous and Endogenous Nitric Oxide on the Airway and Tissue Components of Lung Resistance in the Newborn Piglet

Despite widespread reports of the vasodilatory actions of nitric oxide(NO), little is known of the relaxant effect of NO on newborn airways or lung parenchymal structures. We studied the effects of inhaled NO at 20, 40, and 80 ppm on lung (Rl), tissue (Rti), and airway (Raw) resistance in 13 2-5-d-old anesthetized, ventilated, open-chested piglets. Rl was measured from transpulmonary pressure and air flow. Rti was measured by alveolar capsules, and Raw was calculated as the difference between Rl and Rti. Any given concentration of inhaled NO (20, 40, or 80 ppm) significantly decreased Rl (p< 0.001), Rti (p < 0.001), and Raw (p< 0.05). In addition, blockade of endogenous NO with 30 mg/kg Nω-nitro-L-arginine methyl ester (L-NAME) given i.v. in 12 piglets significantly increased Rti and Rl with variable changes in Raw, and caused a decrease in dynamic compliance. Readministration of NO to eight piglets induced a significant decrease in Rl and Rti at 20 and 80 ppm, whereas Raw significantly decreased only at 80 ppm. Pulmonary arterial pressure decreased after exposure to inhaled NO and increased after L-NAME administration. Systemic arterial pressure was unaffected by inhaled NO but increased after L-NAME administration. Our results indicate that Rl, Raw, and Rti are reduced by exogenous NO, suggesting NO-mediated airway smooth muscle relaxation throughout the newborn lung. In contrast, blockade of endogenous NO significantly increases only Rti, suggesting a physiologic role for endogenous NO in regulation of peripheral contractile elements. We speculate that NO-mediated modulation of resistance in pulmonary parenchyma may serve to regulate the balance of ventilation and perfusion and resultant gas exchange in the lungs during early postnatal development.

α2-Adrenergic receptors are not required for central anti-hypertensive action of moxonidine in mice

In the mouse medulla oblongata, we characterized binding properties and functional responses of two recognition sites for imidazoline compounds: I(1)-imidazoline and alpha(2)-adrenergic receptors. The mouse medulla expresses a higher density of I(1)-receptors than in the rat, whereas alpha(2)-receptor densities were similar between the two species. In anesthetized, ventilated and paralyzed mice, we tested the hypotensive actions of the I(1)/alpha(2) agonist moxonidine, determined its central site of its actions, and the relative roles of I(1) and alpha(2)-receptors. Experiments were performed in C(57)Bl(6) wild type and alpha(2A)-adrenergic receptor deficient mice. In both types of mice, neuronal activation within the rostral ventrolateral medulla (RVLM) region by glutamate microinjection elicited increases in arterial pressure. Moxonidine (0.5 nmol/site/10 nl) microinjected bilaterally into this vasopressor region decreased arterial pressure by 30% and heart rate by 11% in wild type mice. Efaroxan, the I(1)/alpha(2) antagonist (0.4 nmol) when microinjected into the RVLM elevated blood pressure itself and abolished the action of moxonidine, whereas alpha(2)-blockade with SK&F 86466 had no significant effect on blood pressure and did not attenuate moxonidines effect. To more definitively test the role of alpha(2)-adrenergic receptors in the action of moxonidine, moxonidine was microinjected into the RVLM of alpha(2A)-adrenergic deficient mice. The decreases in arterial pressure were nearly identical to those of wild type mice, whereas bradycardia was attenuated. Thus, in the mouse moxonidine acts within the RVLM region to lower arterial pressure mainly through the I(1)-imidazoline receptor independent of alpha(2)-adrenergic receptors.

The excitatory amino acid glutamate mediates reflexly increased tracheal blood flow and airway submucosal gland secretion.

In six decerebrated and in eight alpha-chloralose anesthetized, paralyzed and mechanically ventilated beagle dogs, we have studied involvement of glutamate and glutamate receptors in transmission of excitatory inputs from the airway sensory receptors to the nucleus tractus solitarius and from this site to airway-related vagal preganglionic cells that regulate the tracheal circulation and the submucosal gland secretion. Stimulation of airway sensory fibers by lung deflation-induced reflex increase in tracheal blood flow and submucosal gland secretion. These responses were diminished by prior administration of AMPA/kainate receptor antagonist CNQX into the fourth ventricle (n=6). Furthermore, topical application or microinjection of AMPA/kainate receptor blockers, into the region of the ventrolateral medulla, where airway-related vagal preganglionic neurons are located, abolished the reflex changes in tracheal submucosal gland secretion (n=8); in these dogs mucosal blood flow was not measured). These findings indicate that reflex increase in tracheal blood flow and submucosal gland secretions are mediated mainly via release of glutamate and activation of the AMPA/kainate subtype of glutamate receptors.

CO2-induced c-fos expression in medullary neurons during early development

In this study, we characterized the responses of brainstem neurons to hypercapnic loading at 5, 15, and 40 postnatal days, using c-fos gene encoded protein (Fos), as a marker of neuronal activity. At any of these studied ages exposure to 10% CO2 for 1 h produced a significant increase in the number of activated neurons within the ventral and the dorsal aspects of the brainstem. In the ventrolateral aspect of the medulla oblongata, Fos positive cells were observed within the ventrolateral medulla, extending from the pontomedullary border to the decussation of the pyramids. In the most rostral regions, within the retrotrapezoid field, the number of Fos positive cells was lower than in caudal ventral medullary regions at the levels of the area postrema and the caudal to it. No age related differences were observed in the number of neurons exhibiting CO2-induced Fos expression. Fos positive cells were additionally observed in the lateral paragigantocellular and gigantocellular reticular nuclei, in the medullary midline complex, in the raphe pallidus and in the raphe obscurus. The number of activated cells in the midline neurons was higher at 5 than at 40 days of age. In the dorsal aspect of the medulla oblongata Fos positive neurons were observed mainly within the caudal nucleus tractus solitarius (nTS). Postnatal age had no effect on the distribution and number of nTS cells activated by hypercapnic loading. These findings indicate that neurons activated by increases in CO2/H+ concentrations appear to be well developed from the first days of postnatal life in maturing rat pups.

The role of the medullary raphe nuclei in regulation of cholinergic outflow to the airways

In these studies we determined the role of the medullary midline nuclei on a cholinergic outflow to the airways by examining the response of tracheal tone and lung resistance to pharmacological stimulation. Studies were performed on alpha-chloralose-anesthesized, paralyzed and mechanically ventilated cats, and ferrets. L-glutamate microinjection into the medullary midline neurons significantly decreased tracheal tension, and reduced lung resistance. These effects were abolished by prior topical application of methysergide, a broad spectrum serotonin receptor antagonist. Stimulation of the medullary raphe nuclei was also associated with a significant increase in the phrenic nerve output, and a decrease in arterial blood pressure. The results indicate that the medullary midline neurons are involved in regulation of cholinergic outflow to the airways, and raise the possibility that alterations in the serotonergic pathways may cause airway dysfunction.

Mechanism for substance P-induced relaxation of precontracted airway smooth muscle during development.

Release of substance P (SP) from sensory nerve endings of the tracheobronchial system modulates airway smooth muscle contraction and may cause relaxation of precontracted airways. We sought to elucidate the effect of postnatal maturation on SP-induced relaxation of precontracted airways and determine the roles of endogenously generated nitric oxide (NO) and prostaglandins (PGs). Cylindrical airway segments were isolated from the midtrachea of rats at four different ages, 1, 2, and 4 wk and 3 mo, and contracted to 50-75% of the maximum response induced by bethanechol. SP was then administered in the absence and presence of the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME), the PG inhibitor indomethacin, or both. Relaxation of airways with SP decreased significantly with advancing postnatal age. SP-induced tracheal relaxation was consistently attenuated by pretreatment withl-NAME, indomethacin, or both. In a different group of animals,l-NAME significantly attenuated the relaxant response of airways to PGE2 exposure, but indomethacin had no significant effect on the relaxant response to exogenous NO. We conclude that SP induces a relaxant effect on precontracted airway smooth muscle, which decreases with advancing age and is mediated via SP-induced release of NO and/or PG.