The expression of two kallikrein gene family members in the rat kidney

The mRNAs for two kallikrein gene family members expressed in the rat kidney have been characterized. One mRNA (PS) has previously been found in the pancreas and submaxillary gland and encodes true kallikrein. The second mRNA (K1) encodes a novel kallikrein-like enzyme expressed in the kidney and submaxillary gland that retains many of the key amino acid residues for the characteristic enzymatic cleavage specificity of kallikrein. Two oligonucleotide hybridization probes specific for the K1 mRNA demonstrate that the K1 mRNA is expressed in the kidney and submaxillary gland, but in none of the other eight tissues known to express one or more members of the rat kallikrein gene family. The K1 mRNA is the dominant kallikrein-related mRNA of the kidney, expressed at roughly 10 times the level of the true kallikrein (PS) mRNA. In the submaxillary gland the K1 mRNA is expressed at roughly one-fourth the level of true kallikrein mRNA.     

Androgen dependence of specific kallikrein gene family members expressed in rat prostate

We have used oligonucleotide probes specific for members of the rat kallikrein/tonin gene family (PS, S1, S2, S3, K1, and P1) to establish which arginyl esteropeptidase (kallikrein-like) genes are expressed in the prostate. We have also compared the expression and androgen dependence of these genes in prostate, submaxillary gland (SMG) and kidney. Only S3 (tonin-like) and P1 (kallikrein-like) are expressed in the prostate, with S3 very much more abundant. Prostatic S3 mRNA disappears after 8 days castration and is restored to intact levels by dihydrotestosterone (DHT) but not estradiol benzoate (EB) for 8 days. Prostate P1 mRNA levels were similarly but not identically affected. All six genes are expressed in the SMG, with PS (true kallikrein) the most abundant. Levels of PS mRNA in SMG are unaffected by castration, DHT, or EB treatment, although mRNA levels of other kallikrein-like (S1, K1, and P1), tonin (S2), and tonin-like (S3) genes fall 40-60% after castration, and are unaffected or partially restored by DHT and/or EB administration. Only PS and K1 are expressed in the kidney, at much lower levels than in the SMG and unaffected by castration or steroids. These studies thus confirm and extend the concept of tissue specificity of arginyl esteropeptidase gene expression, and further demonstrate that the same gene(s) is differentially regulated by androgens in the rat prostate, SMG, and kidney.     

Tissue-specific developmental expression of the kallikrein gene family in the rat

Using a series of gene-specific oligonucleotide probes, we have explored the developmental pattern of expression of six members of the rat kallikrein gene family (PS, S1, S2, S3, K1, and P1) in the submandibular gland (SMG) and kidney of both sexes, the prostate and testis of the male, and the anterior pituitary gland (AP) of the female rat. PS (true kallikrein) mRNA was detected in early neonatal life in the SMG and kidney of both sexes. K1, a second kallikrein gene family member expressed in the adult kidney, had a developmental pattern similar to PS in the kidney. In contrast, tonin (S2), S3, K1, and P1, all of which are expressed in the adult SMG, did not reach detectable SMG mRNA levels until puberty in either the male or female rat. Both S3 and P1, which are expressed in the adult prostate, and the novel P1-like mRNA previously detected in the adult rat testis, first appeared in early puberty. In the female AP, PS mRNA levels were not detected until early puberty and thus exhibited a developmental profile different from that of prolactin. The demonstration that S1, S2, S3, P1, and K1 are not expressed in the SMG or prostate until puberty is consistent with the expression of these genes in these tissues being androgen-regulated; the first appearance of PS mRNA in the female AP in early puberty similarly reflects the estrogen dependence of PS gene expression in this tissue. The presence of PS mRNA levels in the SMG and kidney prior to sexual maturation reflects the androgen independence of PS gene expression and suggests that PS (true kallikrein) may play a constitutive and/or developmental role in SMG or renal physiology.     

Biochemical characterization and substrate specificity of rat prostate kallikrein (S3): comparison with tissue kallikrein, tonin and T-kininogenase.

A tissue kallikrein-like enzyme encoded by S3 mRNA was purified to homogeneity from rat prostate gland. The apparent molecular mass of the prostate enzyme is 32 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The intact 32 kDa enzyme is split into two bands of lower molecular mass, 18 and 14 kDa, under reducing conditions on SDS-PAGE. NH2-terminal amino acid sequence analyses of the intact enzyme and heavy and light chains revealed the identity to the translated sequence of a prostate kallikrein cDNA (S3). Isoelectric focusing indicated that the prostate enzyme is a basic protein with pI of 7.30-7.45. Specific activities of the prostate kallikrein toward angiotensin I, angiotensinogen and rat low M(r) kininogen as well as tripeptide chromogenic substrates were compared with those of tissue kallikrein, tonin and T-kininogenase. The kinin-releasing activity is inhibited by leupeptin, antipain, benzamidine and soybean trypsin inhibitor. A sensitive and specific radioimmunoassay for the rat prostate kallikrein shows that the immunoreactive kallikrein levels in prostate and submandibular gland were 23.78 +/- 2.62 micrograms/mg protein (n = 5) and 12.29 +/- 2.25 micrograms/mg protein (n = 5), respectively. The results indicate that the prostate kallikrein S3 is expressed at high levels in both prostate and submandibular glands.     

Oestrogen administration and the expression of the kallikrein gene family in the rat submandibular gland

Using a series of oligonucleotide probes (18-21 mers) specific for members of the rat kallikrein/tonin (arginyl-esteropeptidase) gene family (PS, S1, S2, S3, K1, P1), we have shown by Northern blot analysis that all six genes are expressed in the submandibular gland (SMG), with PS (true kallikrein) the most abundant in both male and female rats. Though female levels of PS mRNA are similar to that in the male, levels of mRNA from both the kallikrein-like (S1, K1, P1) and tonin (S2)/tonin-like (S3) genes are all substantially lower in the female than in the male rat. In contrast with the oestrogen dependence of anterior pituitary kallikrein (PS) gene expression, oestrogen administration (6 micrograms/day for 8 days) to castrate male or female rats is without effect on PS or S1, S2, S3, K1, P1 mRNA levels in the SMG. These findings suggest a tissue-specificity in the oestrogen regulation of true kallikrein gene expression in the two tissues. In intact male rats, oestrogen administration lowers SMG levels of S1, S2, S3, K1, and P1 but not PS mRNA to castrate levels, presumably by suppression of the pituitary/gonadal axis, consistent with the previously reported androgen dependence of SMG expression of these genes with the exception of PS.     

Effect in vivo of beta-adrenergic stimulation, angiotensin II, dibutyryl cyclic AMP, and theophylline on tonin concentration in rat saliva and submaxillary gland.

Tonin (an enzyme present in rat submaxillary gland and saliva) has previously been shown to be able, unlike renin and reninlike substances, to release angiotensin II either directly by acting on an appropriate substrate or from angiotensin I. The administration of a beta-adrenergic drug, isoproterenol, produces a rise of tonin concentration in saliva without affecting its concentration in the submaxillary gland. Prior administration of a beta blocker, propranolol, partially prevents this effect. The administration of theophylline increases the tonin concentration in both saliva and the submaxillary gland, whereas dibutyryl cyclic AMP increases tonin concentration in the former. These results suggest that beta-adrenergic stimulation enhances both tonin release into the saliva and tonin synthesis in the submaxillary gland, and that these effects might be mediated by cyclic AMP. Infusion of angiotensin II blocked the stimulatory effect of isoproterenol on salivary tonin. 1Sar-8Ile-angiotensin II is both a weak antagonist of angiotensin II in this respect and a strong agonist in terms of blocking the effect of isoproterenol another role mirrored in other physiological mechanisms of derivatives of angiotensin II.     

Immunohistochemical localization of tonin and its relation to kallikrein in rat salivary glands

Tonin and kallikrein are serine proteases present in high concentrations in the submandibular gland of the rat. These enzymes release the vasoactive peptides angiotensin II and lysyl-bradykinin from the precursors angiotensinogen and kininogen, respectively. Tonin and kallikrein were purified from homogenates of rat submandibular gland, and antisera against each protein were raised in rabbits. The anti-kallikrein antibody also reacted with tonin, showing partial cross-reactivity between kallikrein and tonin when tested by double immunodiffusion and by immunoelectrophoresis. The anti-tonin antibody did not appear to react with kallikrein in immunodiffusion systems. The cellular localization of tonin was investigated by the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. In the granular tubular cells tonin-specific staining was abundantly present with a granular distribution; in the striated duct cells tonin-specific staining was observed as a thin luminal rim. Tonin was not detected in any other structures of the gland. When the localization of tonin was compared with that of kallikrein, both enzymes were found within the same granular tubular cells. However, more kallikrein than tonin was detected in the striated duct cells. Furthermore, kallikrein but not tonin was found in the ductal cells of the parotid and sublingual glands.