Ingegerd M. Keith

5-HT5a receptors in the carotid body chemoreception pathway of rat.

By using a specific antibody, 5-HT5a receptor-like immunoreactivity was revealed in the chemoreceptive, oxygen sensitive, carotid body (CB) type I cells, and neurons of the petrosal ganglion (PG) and the superior cervical ganglion (SCG) in rat. mRNA encoding for the 5-HTa receptor was also detected in these tissues by RT-PCR, and confirmed with DNA sequencing. The present study provides direct evidence that 5-HT5a receptors are expressed in the CB, PG and SCG, which all likely play fundamental roles in arterial chemoreception.

A guinea pig model for study of bladder mast cell function : histamine release and smooth muscle contraction

To study the function of mast cells in bladder tissue, guinea pigs were sensitized with ovalbumin by intraperitoneal injections, bladder tissue strips were superfused, and tissue contractile force and histamine release were studied. Upon challenge with ovalbumin, bladder tissue contracted 64 +/- 4% (mean +/- S.E.M.) of the maximum carbachol contraction and released 14.1 +/- 1.6% of the total tissue histamine content. Incubation of sensitized bladder tissue with indomethacin led to an increased force and duration of the contraction while incubation with nordihydroguaiaretic acid combined with pyrilamine reduced histamine release and abolished the contraction. Tissue histamine content was significantly higher in the bladder neck than in the dome, and significantly elevated following sensitization. Histochemical studies of bladder tissue demonstrated mast cell degranulation in antigen challenge experiments. In addition, a group of guinea pigs were sensitized to ovalbumin through bladder instillations. With this model, study of the functional characteristics of bladder mast cells and the acute actions of mast cell products on the bladder microenvironment, should now be feasible.

Calcitonin gene-related peptide in hamster lung and its coexistence with serotonin: a chemical and immunocytochemical study

The mammalian lung may have an important endocrine function besides being involved in gas exchange mechanisms. A number of peptide hormones have been localized to neurons and endocrine cells in the lung where they may contribute to the regulation of local pulmonary functions. We have investigated the presence of calcitonin gene-related peptide (CGRP), in the hamster lung by radioimmunoassay and by immunocytochemistry. Measurable quantities of CGRP were detected in lung tissue. Females had higher lung tissue levels of CGRP-like immunoreactivity (IR) than males. This was not reflected in an observable increase in the intensity or distribution of CGRP-like reactivity with immunocytochemistry. Distinct CGRP-like IR was recorded in clustered (NEB) and solitary (NEC) neuroendocrine cells in neonates, weanlings and adults, including all airways from trachea (NEC only) to bronchi, bronchioles, and alveolar ducts to the level of alveoli (NEC and NEB). In adult hamsters, there seemed to be fewer immunoreactive cells, although intensity was unchanged. In addition some NEB contained serotonin-like IR, and colocalization of the peptide and the amine was noted within some cells. Intra-epithelial beaded nerve fibers, subepithelial fibers, and large-caliber nerves in the hilus region and tracheal wall were also CGRP-IR, and immunoreactive nerves were occasionally found in close association with NEB at the basal pole. Positive nerve fibers were not observed in vessels within the lung, and were sparse in the adventitia of tracheal arteries.

Sensory CGRP depletion by capsaicin exacerbates hypoxia-induced pulmonary hypertension in rats

Pulmonary hypertension is a debilitating disease that occurs among infants and adults. One of many etiologies is airway hypoxia. We previously demonstrated a role of endogenous calcitonin gene-related peptide (CGRP), a potent vasodilator, in ameliorating the pulmonary vascular pressor response to chronic hypoxia and related changes in the lungs and heart. This study evaluates the role of endogenous sensory CGRP in hypoxic pulmonary hypertension and examines the intrinsic neural microcircuitry. Rats were pretreated with capsaicin i.p. to deplete pulmonary sensory C-fiber stores of CGRP and substance P and placed in hypobaric hypoxia (10% O2, 16 days) or normoxia together with sham controls. Hypoxia increased pulmonary artery pressure, right-ventricular weight, arterial medial thickness, elasticized capillaries, endothelial cell density, lung water and hematocrit in control rats. Capsaicin augmented pulmonary artery pressure and right-ventricular hypertrophy in hypoxia, and medial thickness and endothelial cell density both in normoxia and hypoxia. Because of the limited effects on these parameters by substance P and other capsaicin-sensitive lung agents, our results suggest that sensory CGRP deficit severely exacerbates pathological signs of hypoxic pulmonary hypertension. A neural microcircuitry consistent with an axon reflex pathway is outlined histochemically. We conclude that endogenous CGRP modulates pulmonary vascular tone in hypoxic pulmonary hypertension which requires intact primary sensory fibers.

Cell relationship in a Wistar rat model of spontaneous prostatitis

PURPOSE Prostatitis in men is a painful, noninfectious inflammatory condition. It is similar to interstitial cystitis which is associated with increased bladder mast cell and sensory nerve fiber density as well as suprapubic pain. Certain strains of rats may provide a useful model for studies of the development of spontaneous prostatitis. We evaluated the time course, and involvement of mast cells and sensory nerve fibers in this process using Wistar rats. MATERIALS AND METHODS The prostates of 4, 6, 8, 10 and 13-week-old male Wistar rats were examined for the degree of inflammation, innervation, mast cell density and nerve mast cell relationship using histochemical and immunocytochemical studies. Bacterial cultures of tissue were performed at 13 weeks. RESULTS The inflammatory cell index increased progressively with age. Inflammation was moderate and consisted mostly of lymphocytes and macrophages associated with occasional glandular epithelial necrosis and edema. The density of nerve fibers immunoreacting with the neuronal marker protein gene produce 9.5 increased gradually with age and fibers immuno-positive for the sensory neuropeptide calcitonin gene-related peptide more than doubled by 13 weeks compared with by 4 weeks. The density of visible mast cells declined after 4 weeks in a pattern that corresponded with the increased percent of mast cells undergoing degranulation. For the mast cells with calcitonin gene-related peptide immuno-positive nerve fibers within a distance of 40 microm. distance correlated significantly with the degree of degranulation. Bacterial cultures were negative at 13 weeks. CONCLUSIONS Our results confirm previous reports of spontaneous prostatitis in Wistar rats and indicate that moderate inflammation may occur in 80% of rats at as early as age 13 weeks. While the correlation of the nerve mast cell axis with mast cell degranulation does not prove our hypothesis of mast cell mediated inflammatory mediator release in the development of nonbacterial prostatitis, it suggests that such a relationship is possible.

In vitro effects of bladder mucosa and an enkephalinase inhibitor on tachykinin induced contractility of the dog bladder

Tachykinin-induced contractility of smooth muscle strips from dog bladders was studied in vitro, and the presence of substance P-like immunoreactivity and neurokinin A and neurokinin B-like immunoreactivity was examined in bladder sections. Nerve fibers with substance P-like immunoreactivity were present in the mucosa, submucosa and smooth muscle. Fibers were also found in nerves, intramural ganglia, and around blood vessels. Neurokinin A-like immunoreactivity had similar distribution, and no neurokinin B-like immunoreactivity was observed. Removal of the mucosa significantly enhanced the sensitivity and the maximum responses to the tachykinins. After removing the mucosa, the sensitivity to these tachykinins increased 0.4 to 0.5 log units (p less than 0.02). The responses to carbachol were not altered by mucosa removal. The leftward shifts of the concentration-response curves for neurokinin A were of similar magnitude after removal of the mucosa, and after pretreatment with phosphoramidon (10 microM), an enkephalinase inhibitor, in the presence of mucosa. However, phosphoramidon did not alter the sensitivity of the bladder strips to neurokinin B, and slightly changed the sensitivity to substance P (0.2 log units). Additional shifts of the substance P and neurokinin A curves to the left were observed in the presence of phosphoramidon when the mucosa was removed (0.6 and 0.5 log units, p less than 0.005). The order of potency for the tachykinins (neurokinin A greater than substance P) was not altered by mucosa removal, addition of phosphoramidon, or both. Neurokinin A was degraded by enkephalinase located in the bladder mucosa and addition of phosphoramidon or mucosa removal resulted in an inhibition or loss of enkephalinase activity. It is concluded that the responses to neurokinin A, which acts on NK-2 type of receptors, prevail on the dog bladder.

PYY-like material and its spatial relationship with NPY, CGRP and 5-HT in the lung of the Syrian golden hamster

SummaryWe investigated the presence of peptide YY, neuropeptide Y, calcitonin gene-related peptide and serotonin in the hamster lung by radioimmunoassay, high performance liquid chromatography and immunocytochemistry. Lung-tissue concentrations of peptide YY and neuropeptide Y were 1.3±0.2 and 2.5±0.2 pmol/g wet weight, respectively. These two closely related pancreatic peptides were demonstrated in separate peaks with high performance liquid chromatography. The peptide YY appeared fragmented as immunoreactive peptide YY eluted primarily late in the gradient but showed additional peaks early in the gradient. Peptide YY-like immunoreactivity (PYY-LI) was predominantly observed in one or more cells of neuroepithelial bodies in all airways peripheral to bronchioles, and in solitary neuroendocrine cells primarily located in the same peripheral areas. Neuropeptide Y-LI was seen in individual, thin nerve fibers around arteries and veins, in the airway lamina propria, and in the airway epithelium; in the latter also immunopositive nerve terminals were located. This pattern did not appear to coincide with that of calcitonin gene-related peptide-LI in epithelial nerve fibers and terminals. Peptide YY-LI, calcitonin gene-related-LI and serotonin-LI were present in cells of one and the same neuroepithelial body. However, peptide YY-LI was never found to be co-localized with calcitonin gene-related-LI or serotonin-LI, but the latter two were co-localized as previously reported.

Specific N-terminal CGRP fragments mitigate chronic hypoxic pulmonary hypertension in rats.

Chronic hypoxic pulmonary hypertension (HPH) is characterized by elevated pulmonary arterial pressure (P(PA)), right ventricular hypertrophy (RVH), pulmonary vascular remodeling, pulmonary edema and polycythemia. Currently, there is no safe and effective treatment for HPH. Calcitonin gene-related peptide (CGRP) is the most potent peptide vasodilator discovered thus far. We previously demonstrated that exogenous CGRP reversed HPH in rats. However, the CGRP1 receptor antagonist CGRP(8-37) and smaller inhibitory C-terminal CGRP fragments that can be formed by enzymatic cleavage in vivo may compromise the beneficial effects of endogenous or exogenous CGRP. We here examine the agonistic efficacy of N-terminal rat alpha-CGRP peptides containing the disulfide bridge (Cys(2)-Cys(7)) with amidated C-terminal in prevention of HPH. Chronic infusion of CGRP(1-8), CGRP(1-13), or CGRP(1-14) at 7 nmol/h/rat via the right jugular vein during 14 days of hypobaric hypoxia (10% inspired O(2)) significantly decreased the P(PA), RVH and pulmonary arterial medial thickness in comparison with controls, suggesting that these CGRP sequences can mitigate chronic HPH in rats. Systemic pressure was unchanged by infused peptides indicating no carry-over effect. In conclusion, N-terminal CGRP fragments (CGRP(1-8), CGRP(1-13) and CGRP(1-14)) may have a protective role in hypoxic pulmonary hypertension.

Expression of 5-HT3 receptors in primary sensory neurons of the petrosal ganglion of adult rats.

By using a specific antiserum, expression of the 5-HT3 receptor was examined in the petrosal ganglion (PG) of adult male rats. We found that the 5-HT3 receptors are widely distributed in the PG. This finding was confirmed by RT-PCR detection of the 5-HT3 receptor mRNA in the tissue. Unlike the distribution patterns of tyrosine hydroxylase (TH), which occurred in limited regions of PG, the 5-HT3 receptors seemed to distribute throughout the ganglion. As many TH-positive neurons in PG innervate type I cells in the carotid body, the coexistence of 5-HT3 receptor and TH in some neurons suggests that this receptor may play a role in carotid body chemoreception.

Continuous, but not Episodic Hypoxia, Induces Creb Phosphorylation in Rat Carotid Body Type I Cells

The CB sensitivity to hypoxia is increased following sustained, long-term hypoxia such as in ventilatory acclimatization to high altitude (Bisgard 1994). The mechanisms underlying the cellular responses and adaptation to hypoxia are poorly understood. It is assumed that a major component of this adaptation is regulation of gene expression. cAMP response element-binding protein (CREB) is a nuclear protein. CREB is activated upon phosphorylation at Ser and modulates the transcription of genes with cAMP response elements (CRE) in their promoters (Ginty 1997). Recently, it has been shown that hypoxia induces phosphorylation of CREB at Ser in cultured PC 12 cells (Beitner-Johnson and Millhorn 1998), which are similar to the CB type I cells in several important characteristics related to oxygen-dependent gene regulation.