D E Van Orden

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.

Role of prostaglandins in estrogen-induced uterine hyperemia

Abstract Estrogen-induced uterine hyperemia is known to result from some other mechanism than direct vasodilatation produced by the hormone. Prostaglandin E 1 has been shown to exhibit properties consistent with a role as a vasodilator intermediate in estrogen-induced hyperemia. In the present study performed on rats the hyperemic response to estrogen as estimated by changes in uterine blood volume was blocked by two different prostaglandin synthesis inhibitors, indomethacin and meclofenamic acid. There was a parallel block of the increase in uterine PGE and PGF activity (radioimmunoassay) produced by estrogen. These results demonstrate that estrogen induces formation of uterine prostaglandins which appear to play a role in promoting the increase in blood supply to the uterus.

Immunocytochemical localization of dopamine-β-hydroxylase in adrenal chromaffin granules

Abstract Electron microscopic localization of dopamine-β-hydroxylase (DBH) was carried out in bovine adrenal medullary chromaffin granule fractions and in intact bovine adrenal medulla by means of a peroxidase-antiperoxidase unlabelled antibody technique according to Sternberger , Hardy , Cuculis and Meyer (1970). Adrenal medullary chromaffin granule fractions showed retention of DBH despite successive fractionation, fixation and prolonged washing procedures, indicating that a portion of the DBH in the chromaffin granule is quite tightly bound. Under these circumstances the electron-opaque granular matrix was absent, implying loss of the more soluble constituents of the chromaffin granules, including matrix-associated DBH. Intact bovine adrenal medulla which was fixed, sectioned and subjected to immunocytochemical localization of DBH showed the enzyme to be present throughout the matrix of the chromaffin granule and demonstrated that, under these less vigorous conditions, the electron-opaque granule matrix was not removed. It would therefore appear to be feasible to differentiate directly between the “soluble” and “bound” forms of DBH by immunocytochemical procedures and to apply this technique to elucidate the process of exocytosis in adrenal medulla and adrenergic nerve terminals.

Effect of prostacyclin inhibition by tranylcypromine on uterine 6-keto-PGF1α levels during estrogen hyperemia in rats

A role for prostacyclin (PGI2) as a mediator of estrogen-induced increases in uterine blood volume (UBV) was investigated by measuring uterine tissue levels of 6-keto-prostaglandin F1 alpha (6-keto-PGF 1 alpha), and testing estrogen responses in rats pretreated with the PGI2 synthesis inhibitor, tranylcypromine (TCP). Uterine 6-keto-PGF1 alpha content was determined by radioimmunoassay of tissue extracts purified through the use of high-performance liquid chromatography (HPLC). Estrogen treatment of castrate rats resulted in a significant increase of uterine 6-keto-PGF 1 alpha was compared to saline treated controls (9.3 ng/uterine horn vs 6.7 ng/uterine horn, p=0.01). Pretreatment with TCP (20 mg/kg) markedly reduced the uterine content of 6-keto-PGF 1 alpha (2.5 ng/uterine horn). The typical 50% increase in UBV observed after estrogen was unaffected by tranylcypromine pretreatment. It was concluded that the increased PGI2 synthesis, as indicated by elevated levels of 6-keto-PGF1 alpha, may function as an amplifying mechanism for the uterine vasodilation-induced by estrogen in castrate rats, but that production of this prostanoid is not essential for the estrogen response.

A technique for the immunohistochemical localization of prostaglandin E.

Prostaglandin E (PGE) has been localized via the unlabeled antibody technique in freeze-dried and ethanol-fixed cryostat sections. Discrete perivascular and stromal localization was present in the uterus prepared by the method presented, but not in classically fixed specimens. Absorption of the anti-PGE by addition of free PGE was ineffective; whereas, removal of PGE-reactive antibodies from the anti-serum was effectively accomplished with an Affigel-101-PGE immunoadsorbant column.

Uterine Serotonin and Receptor Blockade during Estrogen-Induced Uterine Hyperemia

Abstract A role for serotonin in regulation of uterine blood volume (UBV) was investigated by testing estrogen-induced UBV changes in the presence of cyproheptadine and by measuring uterine serotonin concentration at 0, 65, and 125 min following estradiol administration. Rats, castrated on Day 0, were given maintenance doses of estradiol benzoate on Day 7 and 17β-estradiol, 0.5 μg/kg, iv, at time zero on Day 14. At 125 min doses of cyproheptadine which caused a 70% inhibition of the pressor responses to administered serotonin caused a significant reduction in UBV of estrogen-treated animals. Uterine serotonin content was determined by high-performance liquid chromatography with electrochemical detection. Uteri taken at 65 min after the intravenous estradiol showed no significant change in serotonin content or blood volume. At 125 min when the UBV of estrogen-treated animals was 172% of saline control UBV, uterine serotonin was significantly increased (541 ng/g vs 248 ng/g, P = 0.05).

Estrogen-Induced Uterine Hyperemia and Edema Persist during Histamine Receptor Blockade

Summary The effect of H1 and H2 hista-mine receptor antagonists on estrogen-induced increases in uterine blood volume and uterine water content (edema) was determined. Neither the H1 or H2 histamine receptor antagonists nor the combination of both significantly altered either estrogen-induced increases in uterine blood volume or uterine water content. These results suggest that histamine does not play a major role in these physiological responses of the uterus to estrogen.