Deshandra M. Raidoo

Kinin receptors in human vascular tissue: their role in atheromatous disease.

Using samples of many human blood vessels, obtained at autopsy and specific antibodies directed to peptide sequences of the kinin B1 and B2 receptors, we demonstrate the localisation of these receptors within the human vascular system using standard immunolabelling techniques. In large elastic arteries and veins, kinin receptors are present only in the endothelial cells whereas in all muscular arteries and arterioles, these receptors are present in both the endothelial and smooth muscle cells. The identification of kinin receptors in human blood vessels confirms that kinins may modulate both vascular permeability and contractility. The incidental finding at histology, of patchy atheromatous disease in the coronary, femoral, vertebral and pericallosal arteries, assisted in elucidating the role of these receptors in the commonest disease affecting human blood vessels. Intense labelling for B1 receptors was observed in the endothelial cells, foamy macrophages, inflammatory cells and fibroblasts within the thickened intima of the plaque as well as in smooth muscle cells of the underlying tunica media. Immunoreactive B2 receptors were also observed in these cells but with reduced intensity. The intense immunolabelling of B1 receptors in these regions suggest that these may be induced by atheromatous disease and may have therapeutic importance for the B1 receptor antagonists.

Experimental evaluation of collagen sponge as a dural graft

Early collagen products, when used as dural substitutes, promoted severe inflammatory responses and fell into disrepute. A more recent advance, collagen sponge, which is derived from bovine flexor tendons was used in this experimental study. Collagen sponge was surgically implanted as an onlay dural replacement graft following skull trephination and dural excision in 12 primates. Macroscopic, histological and electron-microscopical evaluations were performed at periods of 1, 3 and 9 months. This preliminary animal study indicated that collagen sponge is suitable to use as a graft since it does not induce any inflammatory response or adhesions in the absence of pia arachnoid injury. If forms an ideal scaffold for the early ingrowth of fibroblasts to effect dural repair.

Kinin receptors on human neurones

Knowledge of the distribution of kinin receptors in the human brain will aid our understanding of the role of kinins in neurophysiology. Furthermore, induction of the kinin B1 receptor may be important in the pathogenesis of neural diseases. Using polyclonal antibodies directed to specific regions of the B1 and B2 kinin receptors and standard immunolabelling techniques, we report on the localisation of these receptors on neurones in specific areas of the human brain. B2 bradykinin receptors are present in neurones of the brain stem, basal nuclei, cerebral cortex, thalamus and hypothalamus. B2 immunolabelling was also observed in the endothelial lining of the superior sagittal dural sinus and ependyma of the lateral and third ventricles. B1 kinin receptors have been localised on neurones of the thalamus, spinal cord and hypothalamus. Although binding of labelled bradykinin to neuronal membranes has been demonstrated, this is the first conclusive evidence for the existence of immunoreactive B1, and further confirmation of B2 receptors on human neurones.

Screening of plants used by Southern African traditional healers in the treatment of dysmenorrhoea for prostaglandin-synthesis inhibitors and uterine relaxing activity.

Plants used by Southern African traditional healers for the treatment of menstrual pains were screened for prostaglandin-synthesis inhibitors and the ability to reduce isolated uterine muscle contraction using the cyclooxygenase and in vitro uterine bioassays respectively. Prostaglandins are synthesized from arachidonic acid and the enzyme that drives this reaction is cyclooxygenase. The excessive production of prostaglandins by the myometrium and endometrium induces uterine contractions. Inhibition of cyclooxygenase and hence of the prostaglandin biosynthetic pathway may lead to relief of menstrual pain. Ten plants used by traditional healers for menstrual pains were assayed for cyclooxygenase inhibitory activity. Several plant extracts exhibited high inhibitory activity in the assay. The highest activities were obtained with ethanolic extracts of Siphonochilus aethiopicus, Cenchrus ciliaris and Solanum mauritianum. Generally ethanolic extracts gave higher activity than the aqueous extracts. None of the ethanolic plant extracts were able to relax or reduce the contractions of the precontracted guinea pig uterus.

PATHOPHYSIOLOGY OF THE KALLIKREIN-KININ SYSTEM IN MAMMALIAN NERVOUS TISSUE

The nervous system and peripheral tissues in mammals contain a large number of biologically active peptides and proteases that function as neurotransmitters or neuromodulators in the nervous system, as hormones or cellular mediators in peripheral tissue, and play a role in human neurological diseases. The existence and possible functional relevance of bradykinin and kallidin (the peptides), kallikreins (the proteolytic enzymes), and kininases (the peptidases) in neurophysiology and neuropathological states are discussed in this review. Tissue kallikrein, the major cellular kinin-generating enzyme, has been localised in various areas of the mammalian brain. Functionally, it may assist also in the normal turnover of brain proteins and the processing of peptide-hormones, neurotransmitters, and some of the nerve growth factors that are essential for normal neuronal function and synaptic transmission. A specific class of kininases, peptidases responsible for the rapid degradation of kinins, is considered to be identical to enkephalinase A. Additionally, kinins are known to mediate inflammation, a cardinal feature of which is pain, and the clearest evidence for a primary neuronal role exists so far in the activation by kinins of peripherally located nociceptive receptors on C-fibre terminals that transmit and modulate pain perception. Kinins are also important in vascular homeostasis, the release of excitatory amino acid neurotransmitters, and the modulation of cerebral cellular immunity. The two kinin receptors, B2 and B1, that modulate the cellular actions of kinins have been demonstrated in animal neural tissue, neural cells in culture, and various areas of the human brain. Their localisation in glial tissue and neural centres, important in the regulation of cardiovascular homeostasis and nociception, suggests that the kinin system may play a functional role in the nervous system.

Cellular distribution of the endothelin system in the human brain.

The vasoconstrictor endothelin-1 (ET-1) may also act as a neuropeptide. ET-1 is formed by the catalytic action of endothelin-converting enzyme-1 (ECE-1) on big ET-1 and its cellular actions are mediated via ET(A) and ET(B) receptors. Although localisation of these components in rodent brain has been extensively investigated, no single study has mapped their distribution in human brain. Here we describe the localisation of ET-1 mRNA, ET-1, ECE-1, ET(A) and ET(B) receptors within 24 human brain regions. In situ RT-PCR has previously detected ET-1 mRNA in 22 areas (excluding the post-central gyrus and pineal gland), and ET-1 immunoreactivity was visualised in cells of all regions. Using specific antibodies we have immunolocalised ECE-1 and ET(B) receptors in cells of 24 areas, and ET(A) receptors in nine regions (choroidal epithelial cells, neurones in the diencephalon, hippocampus, amygdaloid, dentate nucleus, Purkinje cells of the cerebellum, flocculo-nodular lobe and vermis). ET-1 mRNA, ET-1, ECE-1 and ET(B) receptors were observed in cortical pyramidal cells, neurones (brainstem, basal nuclei, thalamus, insula and claustrum, limbic region), cells in the anterior pituitary gland; nerve cell processes in the pars nervosa; pinealocytes and choroidal epithelial cells. Only ET-1 mRNA, ET-1, ECE-1, and ET(B) receptors were visualised in cerebral capillary endothelial cells. The presence of ET-1 mRNA, ECE-1 and ET-1 in 22 brain regions confirms ET expression and processing in human brain. The localisation of ET-1 and ET(B) receptors suggests receptor-mediated action akin to a neurotransmitter role for ET-1.

Kinin receptors are expressed in human astrocytic tumour cells.

Tissue kallikrein (TK) is known to be present in several tumours in which increased KLK1 (TK) gene expression has been demonstrated. By degrading components of the extracellular matrix, TK may facilitate tumour proliferation and invasion. The vasodilatory effect of the bioactive kinin peptides causes an increase in vascular permeability, thereby enhancing metastasis. Since kinins act by receptor-linked signal transduction mechanisms, the aim of this study was to elucidate the localization and expression of kinin B1 and B2 receptors in surgical samples of human astrocytic tumours. Tumour tissue collected was processed for light, confocal and electron microscopy (EM) and RNA extraction. The mean high intensity of immunolabeling in tumour cells was quantified in pixels per square micrometer using the Analysis 2.1 Prosystem (Soft-Imaging Software, Germany, 1996). The ultrastructural localization of B1 and B2 kinin receptors was performed on ultrathin sections of the resin-embedded tissue, using immunogold-labeled probes. In the human brain, immunoreactive B2 occurs in cortical neurones but not in glial cells, and immunolabeling for B1 receptors is absent in cortical areas. In the present study, in all of the tumours studied so far, immunolabeling for B2 (28.42 pixels/microm2, n = 12) and B1 (14.07 pixels/ microm2, n = 10) was observed on the astrocytic cells. Immunoreactive kinin receptors were also present in endothelial cells of the stromal blood vessels. At EM, the average number of immunogold particles was 14 for B2 receptors and eight for B1 receptors. The immunoreactive B2 receptors were located closer to the periphery of the tumour cells while B1 immunolabeling was observed throughout the cell.

Visualisation of tissue kallikrein and kinin receptors in oesophageal carcinoma.

Gene expression of tissue kallikrein (TK) and the subsequent formation of kinins may stimulate proliferation of tumour cells by increasing vascular permeability, and enhancing metastasis. Our study was undertaken to immunolocalise TK and B2 receptor in specimens of oesophageal carcinoma immediately following oesophagectomy. The diagnosis and grading of oesophageal carcinoma was performed histologically. For localisation of TK, tissue prokallikrein (T proK) and the kinin receptors, slide-mounted tissue sections were subjected to peroxidase-antiperoxidase method and immunofluorescent staining using primary antibodies raised against each antigen. TK was immunolocalised in giant cells of both well and poorly differentiated squamous cell carcinomas (SCCs). The precursor of the enzyme, T proK, was also immunolocalised. The most intense tissue-prokallikrein labelling was observed in giant tumour cells. Kinin receptors were immunolocalised in giant tumour cells, and the mature squamous cells. These findings suggests that TK, tissue-prokallikrein and kinin receptors are present in oesophageal carcinoma, and may be important in the process of tumourogenesis in this disease.

Cellular visualization of tissue prokallikrein in human neutrophils and myelocytes

The vasoactive peptides bradykinin and kallidin (lysyl‐bradykinin) have been implicated in diapedesis, a cellular process by which neutrophils migrate through endothelial cell gap junctions. The kinin peptides are released from their precursor moiety, kininogen, by the specific action of endoproteinases, the kallikreins. Kininogens have been demonstrated on the surface of neutrophils, and the presence of a competent processing enzyme such as tissue prokallikrein in neutrophils has been postulated, but firm evidence for this is still lacking. We have raised antibodies to a synthetic peptide that is a sequence copy of the activation segment of human TK and demonstrated that the anti‐peptide antibodies specifically recognized the zymogen but not the active form of kallikrein. Using these anti‐peptide antibodies, we showed by Western blotting, immunocytochemistry and electron microscopy that the tissue prokallikrein antigen was localized in neutrophils and their precursor cells, the myelocytes. We further demonstrated by in situ hybridization the presence of tissue kallikrein mRNA in the mature neutrophils and myelocytes. Our findings lend credence to the hypothesis that upon release and activation, neutrophil‐borne TK acts on cell‐associated kininogens to trigger the release of kinins, which may open endothelial gates for neutrophil diapedesis.

Immunolocalisation of endothelin-1 in human brain

The potent vasoconstrictor endothelin-1 (ET-1) may function as a neuropeptide and be a contributing factor in some neurological disorders, e.g. Alzheimers dementia. The presence of ET-1 has been studied more extensively in the rat and porcine nervous systems than in the human brain. Also, the recent description of the extensive ET-1 mRNA localisation in human neural tissue supports expression in regions of human brain not previously investigated. Using specific anti-ET-1 polyclonal antiserum, we immunolocalised ET-1 in 24 regions of human brain autopsy tissues, and correlated this with ET-1 mRNA distribution. ET-1 immunoreactivity was observed within some cells of all the 24 areas examined. Neuronal staining for ET-1 was demonstrated within the diencephalon, brainstem, basal nuclei, cerebral cortex, cerebellar hemisphere, amygdala and hippocampus. In addition, ET-1 immunolabelling was visualised in the pituitary gland as well as in the choroid plexus. The primary sensory cortex and pineal gland also contained immunoreactive ET-1, although ET-1 mRNA had never been detected in these regions previously. The localisation of ET-1 and its subsequent correlation with ET-1 mRNA in most of the regions investigated suggest a more extensive distribution of the ET system in the human brain than was previously identified.