- Email: [email protected]
Substance P immunoreactivity in the liver fluke, Fasciola hepatica
363
SUBSTANCE P IMMUNOREACTIVITY
IN THE LIVER FLUKE, Fasciola hepatica
R.M. MAGEE l , W.L. FOY 2 , I. FAIRWEATHER I , C.F. JOHNSTON 2 , D.W. HALTON 1 and K.D. BUCHANAN 2, iDepartment of Zoology and 2Department of Medicine, The Queen's University, Belfast, Northern Ireland Substance P (SP) has a wide phylogenetic distribution, having been demonstrated immunocytochemically in organisms as widely diverse as coelenterates and man. The present study represents the first description of its presence in a helminth parasite, namely, the liver fluke, Fasciola hepatica, a parasite of major agricultural importance. Using the indirect immunofluorescence technique and cryostat sections of adult flukes, SP-like irmnunoreactivity (IR) has been localised in nerve cell bodies and fibres throughout the CNS and PNS. SP-IR has also been identified, quantified and partially characterized in extracts of F. hepatica by means of radioimmunoassay, gel permeation chromatography and high-performance liquid chromatography (HPLC). Acid alcohol extracts and chromatographed fractions were assayed using an antiserum directed towards the C-terminal region of synthetic SP. Extracts were chromatographed using a Sephadex G50 gel permeation column and 0.5 M acetic acid as the mobile phase, and a C18 ~Bondapak reverse phase analytical column. The level of SP-IR in extracts of whole E, hepatica was measured at 688 pg g-I wet weight. Gel permeation chromatography revealed a single peak which co-eluted with synthetic SP and synthetic substance K (SK). HPLC analysis of this material resulted in the resolution of 3 peaks of IR, the first of which co-eluted with SK standard, and the second with SP standard. The third peak (which was the most hydrophobic) may represent neurokinin B-like IR. In conclusion, the study has shown that 3 tachykinins of similar molecular size are present in Fasciola, and that 2 of these substances are similar to SP and SK found in higher organisms.
DISTURBANCES IN N E U R O P E P T I D E y (NPY) AND OTHER HYPOTHALAMIC PEPTIDES IN RODENT MODELS OF DIABETES Z OBESITY
REGULATORY
G WILLIAMS, M H CARDOSO, M A GHATEI, A R DIANI, G C GERRITSEN, J M POLAK, S R BLOOM: DEPTS. OF MEDICINE AND HISTOCNEMISTRY, ROYAL POSTGRADUATE MEDICAL SCHOOL, LONDON, UK; AND UPJOHN COMPANY, KALAMAZOO, MICHIGAN, USA We examined several hypothalamic peptides implicated in appetite and metabolic control in 3 rodent models of diabetes ± obesity. The KKAy mouse spontaneously develops hyperinsulinaemia, hyperphagia and obes.ity (= prediabetic phase), and finally type 2 diabetes. Susceptible Chinese hamster sublines develop hyperphagia and insulin-deficient diabetes. Diabetes was induced in 12-week male Wistar rats by streptozotocin (STZ: 55 mg/kg). Peptides were extracted by boiling the hypothalamus in 0.5 M acetic acid; bombesin, galanin, NPY, neurotensin, somatostatin and VlP were measured by radioimmunoassay. Prediabetic and diabetic KKAy mice showed significant 30-50% reductions in hypothalamic NPY, galanin and VIP, compared with matched non-diabetic C57BL/6 controls. In diabetic hamsters, NPY and somatostatin were significantly 30% lower than in matched non-diabetic. STZ-diabetic rats (n=12 per group) showed significant 30-50% increases in hypothalamic NPY over controls at 4 and 6 weeks of diabetes. Hypothalamic peptide changes may be associated with hyperphagia and/or central adjustment of glucoregulatory hormone secretion in diabetes, and could underlie a postulated primary hypothalamic defect in the KKAy mouse. NPY powerfully stimulates feeding and insulin secretion when injected centrally; disturbances in all 3 models suggest a key role in hypothalamic appetite and metabolic control. As centrally-administered NPY also inhibits gonadotrophin and growth hormone release, NPY changes could also contribute to abnormal pituitary function in diabetes.
SUBSTANCE P IMMUNOREACTIVITY
IN THE LIVER FLUKE, Fasciola hepatica
R.M. MAGEE l , W.L. FOY 2 , I. FAIRWEATHER I , C.F. JOHNSTON 2 , D.W. HALTON 1 and K.D. BUCHANAN 2, iDepartment of Zoology and 2Department of Medicine, The Queen's University, Belfast, Northern Ireland Substance P (SP) has a wide phylogenetic distribution, having been demonstrated immunocytochemically in organisms as widely diverse as coelenterates and man. The present study represents the first description of its presence in a helminth parasite, namely, the liver fluke, Fasciola hepatica, a parasite of major agricultural importance. Using the indirect immunofluorescence technique and cryostat sections of adult flukes, SP-like irmnunoreactivity (IR) has been localised in nerve cell bodies and fibres throughout the CNS and PNS. SP-IR has also been identified, quantified and partially characterized in extracts of F. hepatica by means of radioimmunoassay, gel permeation chromatography and high-performance liquid chromatography (HPLC). Acid alcohol extracts and chromatographed fractions were assayed using an antiserum directed towards the C-terminal region of synthetic SP. Extracts were chromatographed using a Sephadex G50 gel permeation column and 0.5 M acetic acid as the mobile phase, and a C18 ~Bondapak reverse phase analytical column. The level of SP-IR in extracts of whole E, hepatica was measured at 688 pg g-I wet weight. Gel permeation chromatography revealed a single peak which co-eluted with synthetic SP and synthetic substance K (SK). HPLC analysis of this material resulted in the resolution of 3 peaks of IR, the first of which co-eluted with SK standard, and the second with SP standard. The third peak (which was the most hydrophobic) may represent neurokinin B-like IR. In conclusion, the study has shown that 3 tachykinins of similar molecular size are present in Fasciola, and that 2 of these substances are similar to SP and SK found in higher organisms.
DISTURBANCES IN N E U R O P E P T I D E y (NPY) AND OTHER HYPOTHALAMIC PEPTIDES IN RODENT MODELS OF DIABETES Z OBESITY
REGULATORY
G WILLIAMS, M H CARDOSO, M A GHATEI, A R DIANI, G C GERRITSEN, J M POLAK, S R BLOOM: DEPTS. OF MEDICINE AND HISTOCNEMISTRY, ROYAL POSTGRADUATE MEDICAL SCHOOL, LONDON, UK; AND UPJOHN COMPANY, KALAMAZOO, MICHIGAN, USA We examined several hypothalamic peptides implicated in appetite and metabolic control in 3 rodent models of diabetes ± obesity. The KKAy mouse spontaneously develops hyperinsulinaemia, hyperphagia and obes.ity (= prediabetic phase), and finally type 2 diabetes. Susceptible Chinese hamster sublines develop hyperphagia and insulin-deficient diabetes. Diabetes was induced in 12-week male Wistar rats by streptozotocin (STZ: 55 mg/kg). Peptides were extracted by boiling the hypothalamus in 0.5 M acetic acid; bombesin, galanin, NPY, neurotensin, somatostatin and VlP were measured by radioimmunoassay. Prediabetic and diabetic KKAy mice showed significant 30-50% reductions in hypothalamic NPY, galanin and VIP, compared with matched non-diabetic C57BL/6 controls. In diabetic hamsters, NPY and somatostatin were significantly 30% lower than in matched non-diabetic. STZ-diabetic rats (n=12 per group) showed significant 30-50% increases in hypothalamic NPY over controls at 4 and 6 weeks of diabetes. Hypothalamic peptide changes may be associated with hyperphagia and/or central adjustment of glucoregulatory hormone secretion in diabetes, and could underlie a postulated primary hypothalamic defect in the KKAy mouse. NPY powerfully stimulates feeding and insulin secretion when injected centrally; disturbances in all 3 models suggest a key role in hypothalamic appetite and metabolic control. As centrally-administered NPY also inhibits gonadotrophin and growth hormone release, NPY changes could also contribute to abnormal pituitary function in diabetes.