Kallikrein-binding protein levels are reduced in the retinas of streptozotocin-induced diabetic rats.

PURPOSE: To determine the involvement of rat kallikrein-binding protein (RKBP) in the development of diabetic retinopathy. METHODS: Diabetes was induced by streptozotocin (STZ) (55 mg/kg body weight in 0.05 M citrate buffer, pH 4.5) in male Sprague-Dawley rats (150 to 175 g, 6 weeks old) as confirmed by hyperglycemia and reduced body weight. Retinas were dissected from animals at 1, 2, and 4 months of diabetes. The functional activity of RKBP in retinal homogenates was determined by its complex formation with tissue kallikrein. Immunoreactive RKBP levels were measured by enzyme-linked immunosorbent assay. The RKBP messenger RNA (mRNA) levels in the retina were measured by Northern blot analysis using the RKBP complementary DNA probe. The activity of total Na+,K(+)-ATPase was determined by a radioassay. Total protein concentration was determined by a protein assay. RESULTS: The kallikrein-binding activity was reduced in the retinas of STZ-diabetic rats at 1 (59%), 2 (50%), and 4 (38%) months of diabetes compared to those of age-matched control subjects. Levels of immunoreactive RKBP were significantly lower in the diabetic animals at each time point examined compared to those of control subjects. At 1 and 2 months of diabetes, RKBP levels (nanogram/milligram protein) were decreased significantly to 6.9 +/- 0.7 (n = 8) and 10.6 +/- 1.0 (n = 8), respectively, compared to those of age-matched control subjects (14.1 +/- 0.7, n = 8, P < 0.001, and 14.1 +/- 1.2, n = 8, P < 0.01). At 4 months of diabetes, retinal RKBP levels were lower in both control and diabetic groups, but RKBP levels in diabetic groups were significantly lower (5.8 +/- 0.6, n = 8) than those of the age-matched control subjects (8.4 +/- 0.9, n = 8, P < 0.01). Similarly, Northern blot analysis showed that RKBP mRNA levels were reduced in the retina of each group of STZ-diabetic rats, suggesting that the decrease in RKBP occurred at the level of transcription. CONCLUSIONS: The results show that STZ-induced diabetic rats have decreased retinal RKBP; moreover, this suggests that RKBP may contribute to diabetic retinopathy.

Phe-Met-Arg-Phe-amide (FMRFamide) stimulated growth hormone secretion in conscious OVX rats.

The presence of FMRFamide (Phe-Met-Arg-Phe-amide)-like immunoreactivity in neuronal elements in the hypothalamus suggested a role for this in the hypothalamic control of the anterior pituitary function. We report here the action of FMRFamide on growth hormone release following intracerebroventricular administration to rats. The injection of 200 ng (313.8 picomoles) of FMRFamide (in 2 ul) produced a significantly increased plasma GH 15 min after injection. The GH-increasing effect of 400-800 ng (627-1255 picomoles) of FMRFamide was already developed after 5 min and lasted up to 30 min. No change was detected in the plasma FSH, LH and prolactin levels at any time during the experimental period. The intravenous administration of 10, 30 or 100 ug of FMRFamide had no effect on the plasma GH level. We conclude that FMRFamide can act at low doses to increase GH release through the inhibition of somatostatin release or the stimulation of GRF. We could not exclude a direct site of action in the pituitaries.

A possible role of prostacyclin to stimulate prolactin and growth hormone release by hypothalamic and pituitary actions, respectively.

Prostacyclin (PGI2) (1-5 micrograms in 3 microliters 0.05 M Tris/HCl buffer, pH 7.5) and its stable metabolite, 6-oxo-PGF1 alpha, were microinjected into the third ventricle of ovariectomized rats, and plasma FSH, GH, PRL, and TSH levels were measured by RIA. Control animals received 3 microliters buffer. Injection of 5 micrograms PGI2 dramatically elevated plasma PRL values (4- to 5-fold) at 5 and 15 min, whereas the same dose of 6-oxo-PGF1 alpha produced a significant but smaller (2-fold) stimulatory effect. A delayed increase (1.5-fold) in plasma GH occurred after intraventricular PGI2 at 30 and 60 min. 6-Oxo-PGF1 alpha failed to alter GH levels. There were no alterations in plasma FSH and TSH after intraventricular injection of PGI2. Dispersed, overnight cultured cells from anterior pituitaries of ovariectomized rats were tested with 10(-4)-10(-7) M PGI2 and its metabolite. After 15 min of incubation, 3 X 10(-5) PGI2 produced a highly significant elevation in GH release (P less than 0.001), whereas there was no alteration in PRL levels. Only pharmacological doses of 6-oxo-PGF1 alpha (10(-4) M) stimulated GH release. There was no alteration in PRL release by the cultured cells even in the presence of 10(-4) PGI2. These results suggest that PGI2 stimulates PRL release by a hypothalamic action either to increase the release of PRL-releasing factor, or to decrease release of PRL-inhibiting factor, or by both mechanisms. The delayed stimulatory effect of PGI2 on the release of GH may be exerted via an effect on the anterior lobe itself, since PGI2 was effective in stimulating GH release by the incubated pituitary cells.

Regulation of brain prostaglandins by sexual steroids.

Hypothalamic PGF2 alpha content was determined by radioimmunoassay in rats. The male animals had significantly higher PGF/2 alpha level than the females. The hypothalamic PGF2alpha content increased following orchidectomy (GDX), while ovariectomy alone or combined with oestrone (100 microgram/kg or 1mg/kg i.m.) resulted in a significant i.m.) alone was ineffective. Testosterone (1 or 5 mg/kg i.m.) in GDX animals restored the hypothalamic PGF2 alpha content to the control level. The synthesis and breakdown of prostaglandins were also studied in the microsomal and cytosol fraction of brain homogenate. The PGD2 was the main product of the arachidonate cascade in rat brain microsomes. OVX increased the formation of PGD2 and diminished the biosynthesis of PGF2 alpha. Oestrone administration to OVX rats enhanced the formation of PGF2 alpha. Progesterone injection decreased the biosynthesis of PGF2 alpha and PGD2 alpha and PGD2 in OVX animals. Combined administration of progesterone and oestrone to OVX rats failed to restore the PGF2 alpha synthesis. In orchidectomized animals the arachidonate cascade was found to be depressed, further the synthesis of prostaglandins was normalized after testosterone substitution. The inactivation of 3H-PGF2 alpha by rat brain cytosol fraction was negligible.