C. Andrew Powers

Anterior pituitary glandular kallikrein: trypsin activation and estrogen regulation

Discrepant reports exist regarding the presence of glandular kallikrein or other trypsin-like serine proteases in the pituitary. The existence of pituitary kallikreins in latent forms could explain these discrepancies. I report that trypsin treatment of rat anterior pituitary homogenates activates two serine proteases which generate kinins from kininogen and selectively cleave chromogenic substrates for kallikreins. One protease (enzymatically and immunologically resembling glandular kallikrein) and activated 5-fold by trypsin and was 20 times more abundant in female than in male lobes due to hormonal regulation by ovarian estrogens. The second kallikrein (activated 20-fold by trypsin) was unaffected by estrogens. The results demonstrate that rat anterior pituitary kallikreins predominantly exist in latent forms requiring activation for detection. Additionally, glandular kallikrein is a major estrogen-induced protein in the rat anterior pituitary. No other member of this large protease family is known to be regulated by estrogens.

Trypsin activation, partial characterization, and distribution of kallikrein-like and thrombin-like proteases in the neurointermediate lobe of the rat pituitary.

This study examined whether the neurointermediate lobe (NIL) of the rat pituitary contains latent kallikrein‐ and thrombin‐like proteases activated by trypsin. Partial characterization of such proteases was attempted. Also examined were the distribution of proteo‐lytic activity within the NIL and levels in both male and female lobes. NIL homogenates were assayed for proteo‐lytic activity at pH 8.0 before and after incubation with trypsin (10 μg/ml). Trypsin caused a 10‐fold activation of kallikrein‐like activity and a 40‐fold activation of thrombin‐like activity in NIL homogenates. The kallikrein‐like activity was separated into two components using diethylaminoethyl‐Sephadex. The predominant kallikrein‐like protease was a potent kininogenase closely related or identical to glandular kallikrein and was almost exclusively localized to the intermediate lobe. The second kallikrein‐like protease (kallikrein A) was a weak kininogenase sensitive to inhibition by both soybean trypsin inhibitor and aprotinin and was similarly concentrated in both the neural lobe and the intermediate lobe. The thrombin‐like protease was sensitive to inhibition by hirudin (a specific thrombin inhibitor), clotted fibrinogen, and was slightly more concentrated in the neural lobe than in the intermediate lobe. NILs from female rats contained ∼40% less kallikrein activity than NILs from male rats but did not differ in their content of thrombin‐like activity.

Dynamics of estrogen induction of glandular kallikrein in the rat anterior pituitary

Glandular kallikrein has recently been identified as an estrogen-induced protein of the rat anterior pituitary. This study examined the dynamics of the estrogen induction of anterior pituitary glandular kallikrein in the ovariectomized rat. The estrogen induction of uterine dry weight was also examined for purposes of comparison. 17 beta-Estradiol (0.1-100 micrograms/day) produced dose-dependent increases in anterior pituitary glandular kallikrein, with the highest dose producing a 60-fold increase. Time-course studies demonstrated that a lag phase of 2-3 days was required before these estrogen effects on glandular kallikrein became evident, and levels were still rising between 7 and 10 days of treatment. The dynamics of the estrogen induction of glandular kallikrein resembled the estrogen induction of uterine dry weight with regard to estrogen sensitivity and the presence of a lag phase before estrogen-induced increases. However, uterine dry weight responded more rapidly to estrogen than did anterior pituitary glandular kallikrein, and reached a plateau after 5 days of estrogen treatment.

Differential responses of pituitary kallikrein and prolactin to tamoxifen and chlorotrianisene

Glandular kallikrein, a trypsin-like serine protease, and prolactin (PRL) are both estrogen-induced proteins in rat anterior pituitary lactotrophs. The estrogen agonist and antagonist effects of tamoxifen (TAM, a triphenylethylene antiestrogen) and chlorotrianisene (TACE, a triphenylethylene estrogen) on anterior pituitary glandular kallikrein and PRL were examined to see if TAM and TACE differentially affect these estrogen response of lactotrophs after in vivo dosing of rats. TAM and TACE acted as partial agonists on PRL and uterine weight induction. In contrast, on glandular kallikrein induction TAM acted as a pure estrogen antagonist and TACE acted as an almost pure antagonist. The results document that both TAM and TACE exhibit protein-specific estrogen agonist and antagonist efficacies in lactotrophs, with the estrogen induction of glandular kallikrein being particularly sensitive to antagonism by TAM in vivo. The marked antiestrogen character of TACE was surprising since TACE has been classified and clinically used as an estrogen.

Estrogen and Tamoxifen Interplay with T3 in Male Rats: Pharmacologically Distinct Classes of Estrogen Responses Affecting Growth, Bone, and Lipid Metabolism, and Their Relation to Serum GH and IGF-I

Estrogen (E) and T3 regulate gene expression by receptor mechanisms that may enable hormonal interplay affecting growth and metabolism. Prior studies of E and tamoxifen (TM) interplay with T3 in female rats identified a subset of E responses that required T3 for expression and exhibited large agonist responses to TM. In contrast, TM acted more like an antagonist in most T3-independent E responses. This study used male rats to further explore the role of T3 in E effects on growth and metabolism, and the relation of such effects to changes in serum GH and IGF-I. Orchidectomized, hypothyroid rats were treated 6 wk with vehicle, E2 benzoate (E2B), or TM with or without T3. The following parameters were measured: body weight change; tibia length and bone mineral density; heart and kidney weight; food intake and body temperature; serum levels of glucose, cholesterol, triglycerides, GH, and IGF-I; seminal vesicle weight; and anterior pituitary levels of GH, PRL, glandular kallikrein, and total protein. Interplay...

Dopamine receptor blockade increases glandular kallikrein-like activity in the neurointermediate lobe of the rat pituitary

The effect of dopamine receptor blockers on glandular kallikrein-like activity in the neurointermediate lobe of the rat pituitary was examined. Male rats were given daily injections of haloperidol (2.5 mg/kg), perphenazine (5 mg/kg) or sulpiride (60 mg/kg) for 7 days. Homogenates of the neurointermediate lobe were prepared. Latent proteases were activated with trypsin and proteolytic activity was measured at 37 degrees C, pH 8.0 using chromogenic peptide substrates. All three dopamine receptor blockers produced about a 100% increase in glandular kallikrein-like activity. The results suggest that glandular kallikrein-like activity in the neurointermediate lobe is under inhibitory control by dopaminergic mechanisms.

Cellular mechanisms of estrogen-and dopamine-induced control of glandular kallikrein in the anterior pituitary of the rat

Glandular kallikrein (GK, a trypsin-like serine protease) exhibits estrogen induction and dopamine repression in rat pituitary lactotrophs. Steroid induction may reflect primary actions to increase selectively the synthesis of specific proteins, or may be part of broad cellular responses secondary to steroid-induced phenotype transitions. This study examined the cellular mechanisms underlying estrogen and dopaminergic control of lactotroph GK using a quantified immunocytochemical approach. Pituitaries from ovariectomized rats exhibited little GK staining. Estradiol treatment for 10 days produced dose-dependent increases in pituitary mass, the percentage of lactotrophs (indicating lactotroph proliferation) and the percentage of GK-positive cells. Also, GK staining intensity was dependent upon estradiol dose, increasing 4-fold between 5 μg and 50 μg/48 h. Dopamine receptor blockade with haloperidol (2.5 mg/kg/24 h) elicited weak GK immunostaining in 46% of the lactotrophs in the absence of estradiol, and markedly potentiated GK staining intensity elicited with low but not high doses of estradiol. The results suggest that GK induction is a primary estrogen effect, and is not secondary to a phenotype transition: the induction is enhanced by estrogen-induced lactotroph proliferation. Dopaminergic systems strongly inhibit GK induction by low estradiol levels. This dopaminergic modulation may shift the induction of lactotroph GK to physiological events associated with high estradiol levels or low dopaminergic tone.

Activation of a latent kinin-generating proteinase in the porcine anterior pituitary

This study was conducted to determine whether a kinin-generating proteinase (kininogenase) previously described in the porcine anterior pituitary exists in a latent form. Porcine anterior pituitaries were homogenized in 0.25 M sucrose (pH 7.5) and sequentially centrifuged at 1000 X g for 5 min, 1500 X g for 20 min, 10 000 X g for 20 min, and 105 000 X g for 60 min. The various fractions were assayed for their ability to generate kinins from kininogen and cleave H-D-Pro-Phe-Arg-p-nitroanilide (S-2302) before or after various activation procedures. Untreated pituitary fractions had a small amount of proteolytic activity. However, large increases in kininogenase and S-2302 hydrolytic activity were observed in the 105 000 X g pellet after dialysis, or incubation with trypsin. Repeated freezing and thawing, detergents, phospholipase A2, melittin, plasmin, thrombin, urokinase and Factor Xa failed to activate kininogenase activity in the 105 000 X g pellet. However, plasmin produced massive increases in S-2302 hydrolytic activity. The kininogenase and S-2302 hydrolytic activity was sensitive to inhibition by soybean trypsin inhibitor and aprotinin, and had a broad pH optimum between 7 and 9. The data indicate that the porcine anterior pituitary kininogenase largely exists in a latent form. Also, the porcine anterior pituitary appears to contain an additional latent proteinase which can hydrolyze S-2302.