Charles Julian Lindsey

Neuronal connections of the paratrigeminal nucleus: a topographic analysis of neurons projecting to bulbar, pontine and thalamic nuclei related to cardiovascular, respiratory and sensory functions

The paratrigeminal nucleus, which receives sensory input from trigeminal, glossopharyngeal and vagus nerves, has efferent projections to bulbar, pontine and possibly to thalamic structures associated with nociception, thermoregulation and cardiovascular control. Anterograde neuronal tracers were used to study paratrigeminal efferent connections. Labeled terminal fibers, evidencing bilateral efferent paratrigeminal projections were observed in the medial and caudal solitary tract (sol), lateral reticular nucleus (LRt), ambiguus nucleus (Amb), rostroventrolateral reticular nucleus (RVL), while ipsilateral projections were found in the parabrachial (PB) nuclei and ventral portion of the ventral posteromedial thalamic nucleus (VPM). This extends other findings that describe paratrigeminal projections. Retrograde neuronal transport tracers, microinjected in the defined projection areas were used to map distribution of the paratrigeminal neurons originating different efferent connections. Microinjection of latex microspheres containing fluorescein or rhodamine and Fluoro-gold in the ventral VPM, PB, RVL, Amb, LRt and NTS revealed sets of labeled paratrigeminal nucleus neurons respectively organised in a rostral-caudal sequence. The largest extent of the paratrigeminal nucleus (medial portion) contained neurons projecting to the RVL/Amb, structures associated with cardiovascular regulation. The data show a segmented topographical organization of the nucleus, with different sets of neurons within delimited segments, projecting to neuronal structures associated with different functions. This points to a complex and extensive role for the paratrigeminal nucleus in the integration of somatosensory reflexes related to cardiovascular, respiratory and pain mechanisms. The nucleus may act as a medullary relay interposed between sensory afferents and different structures related to homoeostatic functions.

Projections of the paratrigeminal nucleus to the ambiguus, rostroventrolateral and lateral reticular nuclei, and the solitary tract

The paratrigerminal nucleus (Pa5), a constituent of the spinal interstitial system, was linked to the pressor effect caused by bradykinin injected in the dorsal lateral medulla of the rat. The nucleus receives primary afferent sensory fibers contained in branches of the trigeminal, glossopharyngeal and vagus nerves. In this investigation connections of the paratrigeminal nucleus to other medullary structures were studied with the use of retrograde and anterograde neuronal tracers. Fluorescent light microscopy analyses of medullary sections of rats injected with the retrograde transport tracer Fluoro-gold in the nucleus of the solitary tract (NTS) or in the pressor area of the rostral ventrolateral medulla (RVLM) revealed labeled neuronal cell bodies in the ipsi- and contralateral Pa5. FluoroGold microinjections in the caudal ventrolateral medulla (CVLM) did not produce fluorescent labeling of Pa5 neurons. Microinjection of the anterograde transport neuronal tracer biocytin in the Pa5 produced bilateral labeling of the solitary tract (sol). rostroventrolateral reticular nucleus (RVL), ambiguus nucleus (Amb), lateral reticular nucleus (LRt) and ipsilateral parabrachial nuclei, but not the contralateral Pa5. Confocal laser microscopy showed fluorescence labeling of fibers and presumptive terminal varicosities in the NTS, RVL, Amb and LRt. The present findings showing the paratrigeminal nucleus interposed between sensory afferent and stuctures associated to cardiovascular and respiratory functions, suggest that the structure may act as a medullary relay nucleus for sensory stimuli directly connecting primary afferents to structures mediating cardiovascular and respiratory reflexes.

Pressor effect mediated by bradykinin in the paratrigeminal nucleus of the rat

1 The participation of the paratrigeminal nucleus (Pa5) in the pressor response produced by bradykinin in the dorsolateral medulla of rats was investigated. The microinjection of 6 pmol of bradykinin directly over the paratrigeminal nucleus of unanaesthetized rats produced a significant increase in arterial pressure and a moderate increase in heart rate. 2 Bradykinin microinjections in different sites surrounding the Pa5 compromising the external cuneate nucleus, the trigeminal nucleus, the lateral and ventral spinal trigeminal tract and the dorsal trigeminal tract rostral and caudal to the Pa5 did not elicit significant pressor responses. In contrast, microinjections in the paratrigeminal nucleus produced pressor effects. Injections in the dorsolateral medulla directly over the paratrigeminal nucleus produced larger responses than when injections were made in the nucleus. Saline injections in the different nuclei did not produce pressor effects. 3 Neurochemical lesioning of the Pa5, with microinjections of ibotenic acid in the Pa5, abolished the pressor response to bradykinin injected over the lesioned nucleus. The effect was present, however, when bradykinin was injected on the contralateral side to the lesion, over the intact nucleus of the same animal. Pretreatment with capsaicin (injected in the lateral cerebral ventricle), which causes selective degeneration of afferent sensory fibres, did not alter the pressor effect of bradykinin injected over the paratrigeminal nucleus. 4 Dose‐related responses were produced by different concentrations of bradykinin (0.6–1.8 pmol) microinjected over the nucleus. The bradykinin receptor antagonist HOE 140, injected over the paratrigeminal nucleus 30 min earlier, abolished the pressor response caused by bradykinin. 5 Low doses of bradykinin injected in or directly over the paratrigeminal nucleus increased arterial pressure and caused a small increase in heart rate by stimulating kinin receptors of the paratrigeminal nucleus in the dorsolateral medulla of awake and unrestrained rats. The pattern of the response was consistent with that of sympathetic stimulation. The paratrigeminal nucleus, which receives primary afferents and projects to the nucleus tractus solitarii and the rostral ventral lateral medulla, may be positioned as relay nucleus possibly connecting sensory input to structures that regulate blood pressure.

Bradykinin metabolism pathway in the rat pulmonary circulation.

Objective: The contribution made by different enzymes to the degradation of bradykinin in physiological conditions was estimated by examining bradykinin metabolism in rat serum, in the in situ perfused lung and in vivo. Methods: Dose—response curves for the hypotensive effect of intra‐arterially and intravenously injected bradykinin were obtained in unanaesthetized rats. High‐performance liquid chromatography was used to analyse the products of bradykinin breakdown after incubation with rat serum and perfusion through in situ lung preparations. Results: In rat serum, kininase I degraded 34% and kininase II 11% of bradykinin, no evidence for other activities being detected. In the awake rat, D,L‐2‐mercaptomethyl‐3‐guanidino‐ethylthiopropionic acid, an inhibitor of kininase I, did not reduce the percentage of bradykinin inactivation in the pulmonary circulation. In the in situ perfused lung 65% of bradykinin was metabolized and the main products were BK1‐7, BK1‐5 and BK4‐9. Enalaprilat (an inhibitor of kininase II) blocked the formation of BK1‐7 and BK1‐5 and increased the recovery of BK4‐9. &bgr;‐Mercapto‐ethanol, which inhibits aminopeptidase P, and diprotin A, a specific inhibitor of dipeptidylaminopeptidase IV, both reduced the formation of BK4‐9. Diprotin A also allowed the recovery of BK2‐9. Bradykinin degradation and BK4‐9 recovery were not affected by endopeptidase inhibitors. Conclusions: Our results show that the main degradation pathway of bradykinin in the lung is through the action of kininase II at the carboxyl terminus, and sequential cleavage by aminopeptidase P followed by dipeptidylaminopeptidase IV at the amino terminus. The amino‐terminal degradation of bradykinin represents about 38% of the total lung kininase activity.

Bovine papillomavirus type 4 L1 gene transfection in a Drosophila S2 cell expression system: absence of L1 protein expression

The development of a bovine papillomavirus (BPV) vaccine is an outstanding challenge. BPV protein L1 gene transfection in the Drosophila melanogaster S2 cell expression system failed to produce L1 protein notwithstanding correct L1 gene insertion. Severe genetic inbalance in the host cell line, including cytogenetic alterations, may account for the lack of protein expression.

Clastogenic effect of bracken fern (Pteridium aquilinum v. arachnoideum) diet in peripheral lymphocytes of human consumers: preliminary data

Ingestion of bracken fern (Pteridium aquilinum v. arachnoideum) is associated with digestive tract cancer in different regions of Japan, Venezuela and Brazil. In view of reports that dietary bracken fern causes chromosomal instability in cattle, the clastogenic effect of bracken fern was investigated, in a preliminary study, in peripheral lymphocytes obtained from habitual consumers and a control group of non-consumers, which were carefully investigated about cancer history or family cancer history, negative in both cases, using protocols comparable to those previously described in studies in cattle raised on bracken pastures. Cytogenetic analysis showed significant increased levels of chromosomal abnormalities, such as chromatid breaks, in cultured peripheral lymphocytes of the consumer group. There was no correlation with subjects, gender, smoking habits or alcohol consumption, and the only correlation was with prolonged exposure to dietary bracken.

Baroreceptor-sensitive neurons in the rat paratrigeminal nucleus

The paratrigeminal nucleus (Pa5) is a small collection of medullary neurons localized in the dorsal lateral spinal trigeminal tract. Electrophysiological and anatomical studies showed functional Pa5 efferent connections to the rostroventrolateral reticular nucleus (RVL) and the nucleus of the solitary tract (NTS), both well-studied components of the baroreflex arch. Similarly to the NTS, the main site for termination of cardiovascular peripheral afferents, the Pa5 receives primary sensory inputs of glossopharyngeal and vagus nerves, which suggests that the Pa5 may play a role in the baroreceptor reflex modulation. Simultaneous recording from multiple single neurons in 10 freely behaving rats showed that 37% of recorded Pa5 neurons altered firing rates (35% increased and 2% decreased) during the peak arterial blood pressure response to i.v. phenylephrine. Forty two percent of the 84 identified Pa5 baroreceptor-excited neurons showed high correlation to cardiac cycle denoting the synchronous phasicity to fast changes of blood pressure. Autocorrelation analysis revealed that 48 pressure-sensitive and 55 nonpressure-sensitive neurons have periodical activities which were not directly linked to cardiac cycle. We suggest that the Pa5, a yet unknown component of the baroreflex pathway, may relay baroreceptor information to the NTS and by passing other components of the baroreceptor reflex arch, directly to sympathetic premotor neurons in the RVL.

Effects of teprotide, captopril and enalaprilat on arterial wall kininase and angiotensin converting activity.

We have previously shown [1] that the hypotensive action of angiotensin I (ANG I) converting enzyme (ACE) inhibitors is temporally related to a long-lasting inhibition of kininase activity in the arterial wall. More recently, we showed [2] that conversion of ANG I in the perfused mesenteric vascular bed was not inhibited by enalaprilat at concentrations above those which maximally inhibited kininase activity. The present study extends these observations to two other ACE inhibitors and to another vascular bed, the rat hindlimb preparation. Like enalaprilat, captopril (0.06-1.5 μmol/l) or teprotide (0.4-10 μmol/l) did not inhibit the conversion of ANG I in the perfused mesenteric bed, although the response to bradykinin was substantially potentiated, indicating that the ACE inhibitor decreased kininase activity. In the perfused hindlimb preparations, enalaprilat reduced kininase activity without altering the conversion of ANG I. Enalaprilat or captopril administered to rats caused a decrease in mean arterial blood pressure that lasted for over 24 h. In mesenteric preparations taken from animals 24 h after treatment with ACE inhibitors, kininase activity was inhibited whereas converting activity was unchanged. Therefore, the long-lasting hypotensive effect of ACE inhibition is apparently related to a prolonged inhibition of kininase activity in the arterial wall, which is believed to be the target for ACE inhibitor activity.

Cardiovascular responses to sciatic nerve stimulation are blocked by paratrigeminal nucleus lesion

The paratrigeminal nucleus (Pa5) receives primary sensory inputs from the vagus, glossopharyngeal, and trigeminal nerves and has efferent projections to the nucleus of the solitary tract (NTS), rostroventrolateral reticular nucleus (RVL), as well as to the nucleus ambiguus (Amb), lateral reticular (LRt), parabrachial (PB) and ventral posteromedial thalamic (VPM) nuclei, suggesting that it may play a significant role in cardiovascular responses to nociceptive stimuli. The aim of the present study was to evaluate the effects of unilateral lesions of the Pa5 on cardiovascular alterations induced by afferent somatic sensory nerve stimulation (SNS), also known as the somatosympathetic reflex (SSR). Cardiovascular responses were recorded in rats following either sham operation or unilateral lesions of the Pa5 with ibotenic acid. Mean arterial blood pressure (MAP) increased after SNS, which in sham-lesioned animals raised from 95 +/- 4 to 115 +/- 2 mmHg. Ipsilateral Pa5 lesion did not significantly reduce the pressor response to SNS (from 91 +/- 7 to 107 +/- 4 mmHg increase of baseline MAP). On the other hand, contralateral Pa5 lesion significantly reduced the response to SNS (from 99 +/- 5, to 104 +/- 2 mmHg). Sciatic nerve stimulation did not alter heart rate (HR) neither did ipsi- or contralateral Pa5 lesion HR baseline response level. These findings support a crucial role for the Pa5 in cardiovascular regulation, by relaying SSR input evoked by peripheral nerve stimulation.

Identification of serine proteinases with tonin-like activity in the rat submandibular and prostate glands

Two enzymes with tonin-like activity, designated rSMT3 and rSMT4, were purified from rat submandibular glands and another, rPT1, was obtained from the prostate. The three enzyme fractions hydrolysed angiotensin I, angiotensinogen (AG) and synthetic AG(1-14) to form angiotensin II. With angiotensin I as substrate, pH optima were 6.5 for rSMT3, 6.8 for rSMT4 and 7.5 for rPT1. With AG(1-14), the three enzymes had optimal activity at pH 7.5. The three enzymes had negligible activity upon a kallikrein substrate, Ac-Phe-Arg-Nan. The enzymes were inhibited by aprotinin, soybean trypsin inhibitor and phenylmethanesulfonyl fluoride but not by two angiotensin converting enzyme inhibitors, ethylenediaminetetracetic acid or enalaprilat. N-tosyl-L-phenylalanine chloromethyl ketone (1 mM) inhibited rPT1 and rSMT4 but not rSMT3. Molecular weights (SDS-PAGE) were 31,700 for rSMT3, 29,800 for rSMT4 and 28,100 for rPT1. Total activity in the prostate is 150-times lower than in the submandibular gland, where 92% of the tonin activity is related to rSMT4. Physical and chemical properties suggest that rSMT4 is tonin, whereas rSMT3 and rPT1 are tonin-like enzymes which can generate angiotensin II from different substrates.