Carlo Maccarrone

Neuropeptide Y: A putative neurotransmitter

Since its isolation in 1982, neuropeptide Y (NPY) has received considerable interest. This 36 amino acid peptide has been identified widely throughout the central and peripheral nervous systems, and within the autonomic system it appears in close association but not exclusively within catecholamine containing nerves. NPY begins to meet some of the criteria required to be established as a neurotransmitter. Thus, the peptide has been localised exclusively within nerves, and electron microscopy has shown NPY within nerve terminals. High affinity, saturable binding sites for NPY have been demonstrated in rat brain membranes, and the peptide has been reported to be released into the circulation during sympathetic nerve stimulation. The peptide is pharmacologically active both within the central nervous system by altering blood pressure, feeding and anterior pituitary function and in the periphery where NPY acts as a vasoconstrictor.

Differences in regional brain concentrations of neuropeptide Y in spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats

Regional brain concentrations of neuropeptide Y immunoreactivity (NPY) were measured in age-matched Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats using a sensitive and specific radioimmunoassay developed within our laboratory. In 5 of the 9 brain regions examined the SH rats had significantly lower NPY levels compared to the WKY strain. The largest differences occurred within the cortex (-43%), and cervical (-30%) and thoracic spinal cord (-30%), whilst smaller differences were observed in the midbrain (-11%) and medulla oblongata-pons (-18%). The concentrations of NPY in the hypothalamus and hippocampus did not vary between the strains. The SH rats contained significantly greater (+18%) NPY levels in the striatum compared to the WKY rats.

CLONIDINE: UNDERSTANDING ITS DISPOSITION, SITES AND MECHANISM OF ACTION

The hypotensive action of clonidine was discovered serendipitously in 1962 and its potential as an antihypertensive drug recognized. Subsequently it was first marketed as an antihypertensive drug in Germany in 1966 and its high potency as a hypotensive agent was documented by Knobloch & Morr (1966). These workers reported significant lowering of blood pressure with as small an oral dose as 75 pg (approximately 1 pg/kg) which makes clonidine the most potent antihypertensive drug in the pharmacopeia. Over the next 15 years physicians tended to use higher doses of clonidine (300-900 pg per day) and this led to a significant degree of side effects such as sedation, dry mouth and constipation (Jarrott 1984). However, recently there has been a swing back to the use of low dosage regimens such as 75 pg twice a day and a recent extensive study (Briickner et a/. 1987) of 6700 patients with mild to moderate hypertension on this regimen have confirmed the initial study of Knobloch and Morr (1966) that this dose produces satisfactory control of blood pressure with a low incidence of side effects. Although clonidine is known commonly as an antihypertensive drug, experimental and clinical studies have demonstrated that this drug has several effects other than on the cardiovascular system. For example, clonidine has an analgesic action in laboratory animals, particularly against noxious stimuli such as acetic acid-induced writhing (Paalzow 1974; Bentley et a/. 1977; Spaulding et a/. 1979). Clinically, epidural clonidine is an effective analgesic (Tamsen & Gordh 1984; Kalia et al. 1986) and oral clonidine reduces the required dose of fentanyl by 45% to produce satisfactory anaesthetic depth for procedures such as Iaryngoscopy (Ghignone et al. 1986). Another action of clonidine which is seen as a side effect with the use of clonidine as an antihypertensive drug is sedation (Jarrott 1984) although tolerance to sedation develops rapidly. In laboratory animals, this action is seen as a potentiation of hexobarbitone sleeping time (Timmermans et al. 1981), although this does not mean necessarily that clonidine has a barbiturate-like CNS depressant action. Instead, our studies on the effects of chronic infusion of clonidine (10 pg/kg per h for 9 days) on the spontaneous locomotor activity of normotensive Wistar-Kyoto rats showed that the marked reductions in locomotor activity during the 12 h dark phase were due largely to a clonidine-induced increase in overall sleep time. When awake, clonidine-infused rats were as active as saline-

Intrathecal kynurenate reduces arterial pressure, heart rate and baroreceptor-heart rate reflex in conscious rats

In the present study, an excitatory amino acid (EAA) pathway in the spinal cord which maintains sympathetic vasomotor tone in conscious rats has been investigated. To this end, the cardiovascular effects of an intrathecally administered EAA antagonist, kynurenate (KYN), were studied in conscious rats. KYN (0.5 mumol in 10 microliters) caused a dramatic reduction in mean arterial pressure (MAP) and heart rate (HR) that persisted for 2-3 h, and also resulted in extensor paralysis of the hindlimbs. The time courses of fall in MAP and HR and hindlimb paralysis were similar. Baroreceptor-HR reflex activity was also markedly impaired after KYN, suggesting functional diminution of sympathetic outflow at the level of the spinal cord after blockade of EAA receptors by KYN. Xanthurenate, a metabolite of KYN without EAA antagonistic properties, produced negligible effects at the same dose of KYN. While these findings do not identify the putative EAA pathway, they do provide the first demonstration that this system is tonically active in conscious rats.

Differential effects of surgical sympathectomy on rat heart concentrations of neuropeptide Y-immunoreactivity and noradrenaline

The aim of this study was to estimate the proportion of cardiac neuropeptide Y-immunoreactivity (NPY-ir) which is not present in sympathetic neurones innervating the rat heart. The procedure employed was to surgically sympathectomize the heart and then measure the remaining cardiac concentrations of NPY-ir and noradrenaline (NA). Unilateral (left) sympathectomy significantly reduced the levels of NPY-ir and NA in all regions of the heart (by 40-70%) except for the NPY-ir in the right atrium which was unaltered. The effect of bilateral sympathectomy was significantly greater than that of unilateral sympathectomy. Unilateral and bilateral sympathectomy produced similar reductions in the concentrations of NPY-ir and NA in the ventricular tissue. In contrast dissimilar changes were produced in the atrium. Although bilateral sympathectomy almost totally depleted the NA from the right atrium (by 98%), the NPY-ir levels were only reduced by 50%. These results indicate that approximately half the content of NPY in the right atrium is not present in sympathetic noradrenergic neurones. This pool may occur in the previously described intrinsic neurones of the right atrium.

Beta-adrenoceptor selectivity of dobutamine: in vivo and in vitro studies.

We compared the beta-adrenoceptor stimulant actions of dobutamine and (-)-isoprenaline in isolated tissue preparations (atria, trachea, and uterus) from the guinea pig and in chloralose-anaesthetized, vagotomized cats (arterial blood pressure, heart rate, hindlimb perfusion pressure, and soleus muscle contractility). The results obtained in these experiments indicate that, on a dose basis, dobutamine shows little selectivity in producing alpha-, beta 1-, and beta 2-adrenoceptor-mediated effects. In phentolamine-treated cats, reductions in arterial pressure and total peripheral resistance produced by infusions of dobutamine were little affected by the beta 2-adrenoceptor-selective antagonist butoxamine, but were antagonized by atenolol. The rise in cardiac output produced by dobutamine involved increases in both heart rate and stroke volume. There was little indication of a selective inotropic action, a feature that confirmed the results obtained in isolated atrial preparations. The increase in cardiac output appeared to involve both alpha- and beta-receptor-mediated actions, because phentolamine reduced the rise in cardiac output by reducing stroke volume.

Distribution of neuropeptide Y immunoreactivity in the rat basal ganglia: Effects of excitotoxin lesions to caudate-putamen

The distribution of neuropeptide Y-like immunoreactivity (NPY-LI) within the rat basal ganglia was studied using microdissection with a sensitive radioimmunoassay, and excitotoxin lesions were made in an attempt to characterise the neurones containing NPY in this brain area. Immunoreactivity was unevenly distributed in the basal ganglia of control rats, with concentrations in the caudate-putamen (CP) and nucleus accumbens being appreciably higher than those found in the globus pallidus and substantia nigra (SN). Within the CP, immunoreactivity was concentrated in caudal and extreme rostral aspects. N-Methyl-D-aspartate lesions of the rostral CP significantly reduced immunoreactivity in this area, whilst levels in other regions of the basal ganglia were unaffected. Neurones containing NPY-LI are likely to be intrinsic to the CP and do not appear to project to the globus pallidus or SN.

Comparison of neuropeptide Y immunoreactivity in hypothalamic and brainstem nuclei of young and mature spontaneously hypertensive and normotensive Wistar-Kyoto rats

The concentration of neuropeptide Y immunoreactivity (NPY-ir) was measured in 8 hypothalamic and 5 brainstem nuclei of 6- and 14-week-old spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats. Strain differences were observed in 3 hypothalamic nuclei and age-dependent changes occurred in 3 hypothalamic and 2 brainstem nuclei. In both the ventromedial hypothalamic nucleus and locus coeruleus the observed change in NPY-ir with age in SH rats was significantly different to the change observed in the WKY. These strain- and age-related differences in NPY-ir may be of relevance in the development of hypertension in the SH rat.

Age-Related Changes in Neuropeptide Y Immunoreactivity (NPY-ir) in the Cortex and Spinal Cord of Spontaneously Hypertensive (SHR) and Normotensive Wistar-Kyoto (WKY) Rats

The concentrations of neuropeptide Y immunoreactivity (NPY-ir) were measured in the cortex and cervical, thoracic and lumbar spinal cord of 8-, 18- and 31-week-old normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). This peptide was measured using a highly sensitive and specific radio-immunoassay (RIA) developed in our laboratory. The concentrations of NPY-ir in the cortex, cervical and thoracic spinal cord were significantly different between the two strains, with the levels being consistently lower in the SHR strain independent of age. While there was no obvious change in the levels of NPY-ir in the cortex with increasing age, there was a general trend for the levels to fall in all three spinal cord regions. The rate of decrease of NPY-ir in the thoracic spinal cord appeared greater in the SHR compared with the WKY rats. These biochemical differences observed in the cortex and thoracic spinal cord of SHR and WKY rats may be related to the behavioural and blood pressure differences observed in these strains.

Modification of the circadian body temperature rhythm of the spontaneously hypertensive rat during and following cessation of continu3us clonidine infusion

The effects of continuous clonidine infusion (10 micrograms/kg/h for 10 days) and the cessation of this infusion on the circadian body temperature rhythm of the spontaneously hypertensive (SH) rat were examined. This circadian rhythm was blunted significantly during the infusion of clonidine. The fall in body temperature which normally occurs at the onset of each light phase was attenuated during the clonidine infusion, and as such, these rats displayed a relative hyperthermia over the light but not the dark phases. On cessation of infusion (24:00 h), a distinct hyperthermia occurred within the immediate dark phase and the subsequent light phase. The results demonstrate that the circadian control of body temperature is disturbed both during and after continuous clonidine infusion.