Kallikrein Secretion Research Articles

Uterine kallikrein and arterial bradykinin activities and uterine arterial proliferation in response to acute estradiol-17β exposure in ovariectomized ewes

Estradiol-17β (E2) increases kallikrein in rodent and human reproductive tissues. Kallikrein specific activity is increased in the porcine uterus when conceptus E2 is secreted at maternal recognition of pregnancy. When kallikrein acts on kininogen to liberate bradykinin, angiogenic and vasoactive factors are released. The uterus of ovariectomized ewes administered E2 undergoes rapid vascular changes via different patterns of angiogenic and vasoactive factors. Our hypothesis was that E2 would increase the specific activity and protein secretion of tissue kallikrein in endometrial explants culture media (ECM) and ewes exposed to E2 would have uterine arteries that would be more sensitive to the vasodilatory effects of bradykinin. Ovariectomized ewes received 100 mg of E2 implants for 0, 12, 24, or 48 h. After treatment, uterine weights were determined, and caruncles were processed for ECM. Uterine weights and uterine weight per ewe body weight were significantly greater in the 12 and 24 h ewes compared with the 0 h ewes, with the 48 h ewes being similar to the 24 h ewes. There were no statistically significant differences in caruncular tissue kallikrein protein secretion among the treatment groups. There was a tendency (P = 0.09) for duration of E2 exposure to influence tissue kallikrein specific activity where kallikrein activity was greater (P ≤ 0.05) in the 12 and 48 h ewes compared with the 0 h ewes, with 24 h ewes being intermediate (unprotected F test). Uterine arteries from ewes with E2 for 24 and 48 h had more sensitivity to bradykinin, via the bradykinin receptor 2, than uterine arteries from ewes with 0 or 12 h E2 exposure. We fail to reject our hypothesis as E2 did elicit a positive response in tissue kallikrein specific activity and bradykinin response. Further investigations are needed to determine how kallikrein and bradykinin may be involved in vascular remodeling of the ovine uterus.

Paracrine and endocrine regulation of renal K+ secretion.

The seminal studies conducted by Giebisch and coworkers in the 1960s paved the way for understanding the renal mechanisms involved in K+ homeostasis. It was demonstrated that differential handling of K+ in the distal segments of the nephron is crucial for proper K+ balance. Although aldosterone had been classically ascribed as the major ion transport regulator in the distal nephron, thereby contributing to K+ homeostasis, it became clear that aldosterone per se could not explain the ability of the kidney to modulate kaliuresis in both acute and chronic settings. The existence of alternative kaliuretic and antikaliuretic mechanisms was suggested by physiological studies in the 1980s but only gained form and shape with the advent of molecular biology. It is now established that the kidneys recruit several endocrine and paracrine mechanisms for adequate kaliuretic response. These mechanisms include the direct effects of peritubular K+, a gut-kidney regulatory axis sensing dietary K+ levels, the kidney secretion of kallikrein during postprandial periods, the upregulation of angiotensin II receptors in the distal nephron during chronic changes in K+ diet, and the local increase of prostaglandins by low-K+ diet. This review discusses recent advances in the understanding of endocrine and paracrine mechanisms underlying the modulation of K+ secretion and how these mechanisms impact kaliuresis and K+ balance. We also highlight important unknowns about the regulation of renal K+ excretion under physiological circumstances.

Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii was recently been shown to be an effective bio-manufacturing platform for the production of recombinant proteins. The advantage of using C. reinhardtii is that it is fast to grow, inexpensive to culture, and relatively safe. However, the expression of foreign proteins is always low and difficult to purify in C. reinhardtii. Human kallikrein has the potential to be developed into certain drugs, like insulin. Therefore, its biosynthesis is important to drug development. In this study, we synthesized the sg gene, a signal peptide sequence of alkaline phosphatase, and inserted it into a pH124 plasmid, which contains a HSP70A-RBCS2 promoter and a RBCS2 terminator. Then, we inserted the human kallikrein gene klk1 behind the sg sequence to make a pHsgk124 vector. The pHsgk124 were transferred into a cell-wall deficient strain of C. reinhardtii, cc-503, by using the glass bead method. Southern blot analysis showed that sg and klk1 were incorporated into genes of the transgenic C. reinhardtii. RT-PCR analysis showed that it had an active transcription and its expression increased three times under heat stress. Western blot analyses of proteins inside and outside cells (in the culture medium) showed that klk1 was expressed in the cell and the resulting protein was secreted into medium. An enzyme activity assay showed that the recombinant protein had the ability to hydrolyze the specific substrate H-D-Val-Leu-Arg-Pna. In conclusion, we successfully bioengineered C. reinhardtii to produce and secrete human kallikrein protein, which has important biomedical implications.

Therapeutic Targeting of Sunitinib-Induced AR Phosphorylation in Renal Cell Carcinoma.

Androgen receptor (AR) plays a crucial role in the development and progression of prostate cancer. AR expression has also been reported in other solid tumors, including renal cell carcinoma (RCC), but its biological role here remains unclear. Through integrative analysis of a reverse phase protein array, we discovered increased expression of AR in an RCC patient-derived xenograft model of acquired resistance to the receptor tyrosine kinase inhibitor (RTKi) sunitinib. AR expression was increased in RCC cell lines with either acquired or intrinsic sunitinib resistance in vitro An AR signaling gene array profiler indicated elevated levels of AR target genes in sunitinib-resistant cells. Sunitinib-induced AR transcriptional activity was associated with increased phosphorylation of serine 81 (pS81) on AR. Additionally, AR overexpression resulted in acquired sunitinib resistance and the AR antagonist enzalutamide-induced AR degradation and attenuated AR downstream activity in sunitinib-resistant cells, also indicated by decreased secretion of human kallikrein 2. Enzalutamide-induced AR degradation was rescued by either proteasome inhibition or by knockdown of the AR ubiquitin ligase speckle-type POZ protein (SPOP). In vivo treatment with enzalutamide and sunitinib demonstrated that this combination efficiently induced tumor regression in a RCC model following acquired sunitinib resistance. Overall, our results suggest the potential role of AR as a target for therapeutic interventions, in combination with RTKi, to overcome drug resistance in RCC.Significance: These findings highlight the therapeutic potential of targeting the androgen receptor to overcome RCC resistance to receptor tyrosine kinase inhibitors. Cancer Res; 78(11); 2886-96. ©2018 AACR.

Renal (tissue) kallikrein-kinin system in the kidney and novel potential drugs for salt-sensitive hypertension.

A large variety of antihypertensive drugs, such as angiotensin converting enzyme inhibitors, diuretics, and others, are prescribed to hypertensive patients, with good control of the condition. In addition, all individuals are generally believed to be salt sensitive and, thus, severe restriction of salt intake is recommended to all. Nevertheless, the physiological defense mechanisms in the kidney against excess salt intake have not been well clarified. The present review article demonstrated that the renal (tissue) kallikrein-kinin system (KKS) is ideally situated within the nephrons of the kidney, where it functions to inhibit the reabsorption of NaCl through the activation of bradykinin (BK)-B2 receptors localized along the epithelial cells of the collecting ducts (CD). Kinins generated in the CD are immediately inactivated by two kidney-specific kinin-inactivating enzymes (kininases), carboxypeptidase Y-like exopeptidase (CPY), and neutral endopeptidase (NEP). Our work demonstrated that ebelactone B and poststatin are selective inhibitors of these kininases. The reduced secretion of the urinary kallikrein is linked to the development of salt-sensitive hypertension, whereas potassium ions and ATP-sensitive potassium channel blockers ameliorate salt-sensitive hypertension by accelerating the release of renal kallikrein. On the other hand, ebelactone B and poststatin prolong the life of kinins in the CD after excess salt intake, thereby leading to the augmentation of natriuresis and diuresis, and the ensuing suppression of salt-sensitive hypertension. In conclusion, accelerators of the renal kallikrein release and selective renal kininase inhibitors are both novel types of antihypertensive agents that may be useful for treatment of salt-sensitive hypertension.

A Proposal----The Renal Kallikrein-Kinin System as a Compensating System for Salt Accumulation after Excess Salt Intake

Terrestrial mammals, like human, must reserve water and NaCl to counter the effects of dry-environments. Their kidneys need to reabsorb as much water and sodium as possible along the proximal tubules and collecting duct. Thus, excess sodium intake tends to sodium accumulation in the body. The renal (tissue) kallikrein-kinin system (KKS) exists to prevent excessive sodium reabsorption. Renal kallikrein is secreted from the distal connecting tubule cells, which are located just distal to the major reabsorbing system. Kallikrein releases kinins, which inhibit sodium reabsorption along the collecting ducts. The kinins in the tubules are quickly destroyed by kidney-specific destroying enzymes (kininases), carboxypeptidase Y-like exopeptidase and neutral endopeptidase, which differ completely from those in plasma. Ebelactone B and poststatin were discovered as selective inhibitors of urinary kininases. Excess sodium intake causes hypertension, since sodium accumulation in the blood vessel walls increases sensitivity to hypertensive agents. Potassium, and ATP-sensitive potassium channel blockers cause renal kallikrein secretion. In bradykinin B2 receptor gene knockout mice, tissue kallikrein gene knockout mice, and kininogen-deficient rats, the knockout or deficiency does not itself induce hypertension, but these animals are salt-sensitive. Mutant kininogen-deficient (kinin-free) rats on 2% sodium intake quickly develop hypertension. Urinary kininase inhibitors reduce high blood pressure due to sodium accumulation on high sodium intake. Our ample supporting data suggest that the renal KKS prevents sodium accumulation, and that reduced levels of renal kallikrein may cause salt-sensitive hypertension. Keywords: Excess salt intake, The renal kallikrein-kinin system, Potassium, ATP-sensitive potassium channel blockers, Ebelactone B, Poststatin, Neutral endopeptidase, Renal Kallikrein-Kinin System, Renal kallikrein, Y-like exopeptidase, urinary kininases, Mutant kininogen-deficient, kallikrein-kinin system, renin-angiotensin system, systemic blood pressure, angiotensin-converting enzymes, lysyl-bradykinin, bradykinin, kallikrein-secreting cells, Henle's loop, BK-B2 antagonist, carboxypeptidase Y-like exopeptidase, human urinary kininases, epithelial Na channels, Rattus norvegicus, LMW kininogens, cerebrospinal fluid, deoxycorticosterone acetate, Weinberger's group

An ATP-sensitive potassium channel blocker suppresses sodium-induced hypertension through increased secretion of urinary kallikrein

It is suggested that an ATP-sensitive potassium channel blocker suppresses sodium-induced hypertension through increased secretion of urinary kallikrein. We reported that glibenclamide, an ATP-sensitive potassium channel blocker, accelerated dose-dependent secretion of renal kallikrein in sliced kidney cortex and in vivo in rats. In vehicle-treated normal Brown- Norway-Kitasato (nBN-Ki) rats, the administration of glibenclamide increased urinary kallikrein secretion, but changed neither the systolic blood pressure nor the urinary sodium on low (0.3%) NaCl diets. Although on high (8%) NaCl diets, the systolic blood pressure of the nBN-Ki rats administrated glibenclamide was significantly lower (P<0.05). The urinary levels of kallikrein and sodium of the nBN-Ki rats administrated glibenclamide were significantly increased (P<0.05, glibenclamide vs. vehicle). A similar result was obtained with a kidney-selective ATP-sensitive potassium blocker, N,N'-dicyclohexyl-4-morpholinecarboxamidine (U18177), in SD rats. Mutant kininogen-deficient Brown-Norway Katholiek (muBN-Ka) rats fed high (8%) NaCl diets showed an increase in urinary kallikrein levels, but showed neither hypotensive nor natriuretic actions by glibenclamide. A bradykinin B(2) receptor antagonist, 8-[3-[N-(E)-3-(6-acetamidopyridin-3-yl) acryloylglyycyl]-N-methylamino]-2,6-dichlorobenzyloxy]-2-methylquinoline (FR173657), which was administrated to SD rats, together with glibenclamide, abolished the hypotensive and natriuretic effects of glibenclamide in high-sodium (8%NaCl) hypertension, despite an accelerated secretion of urinary kallikrein. Therefore, these results indicate that glibenclamide, an ATP-sensitive potassium channel blocker suppressed sodium-induced hypertension through sodium excretion from the kidney resulting from accelerated secretion of urinary kallikrein.

Genetic and dietary control of plasma tissue kallikrein secretion and urinary kinins exretion in man

Tissue kallikrein is a major kinin-forming enzyme involved in artery and kidney functions. Urinary tissue kallikrein activity (UKLKa) reflects renal tissue kallikrein activity and depends on Na and K intake, and genetic factors, especially the tissue kallikrein-inactivating polymorphism, R53H. The effect of these factors on the level of kinin peptides is, however, not known. Moreover, a circulating form of tissue kallikrein is present in human plasma but its origin and regulation are unknown. We used a crossover study to investigate UKLKa, plasma tissue kallikrein (pTK), and urinary kallidin peptides and metabolites (Uki) in 10 R53H and 30 R53R normotensive male subjects randomly assigned to either a 1-week low Na-high K or a high Na-low K diet. UKLKa was 50-60% lower in R53H than R53R subjects and was increased by the low Na-high K diet. pTK was also 45-55% lower in R53H than R53R subjects and was increased by the low Na-high K diet. Uki was slightly but significantly higher under the low Na-high K than the high Na-low K diet, but did not differ between genotypes. These observations indicate that pTK levels are genetically determined and regulated by Na and K diet, in parallel with UKLKa; this suggests that circulating tissue kallikrein originates mainly from the kidney, and can contribute to circulatory adaptation to dietary ions. Uki is influenced by the Na and K diet, suggesting that kinins participate in renal adaptation to ion intake, but do not quantitatively reflect tissue kallikrein activity in urine, or presumably, in the kidney.

Caveolin-1-Mediated Expression and Secretion of Kallikrein 6 in Colon Cancer Cells

Kallikreins are secreted proteases that may play a functional role and/or serve as a serum biomarker for the presence or progression of certain types of cancers. Kallikrein 6 (KLK6) has been shown to be upregulated in several types of cancers, including colon. The aims of this study were to elucidate pathways that influence KLK6 gene expression and KLK6 protein secretion in the HCT116 human colon cancer cells. Our data indicate a central role for caveolin-1 (CAV-1), the main structural protein of caveolae, in both KLK6 gene expression and protein secretion. Sucrose gradient subcellular fractionation reveals that CAV-1 and KLK6 colocalize to lipid raft domains in the plasma membrane of HCT116 cells. Furthermore, we show that CAV-1, although it does not directly interact with the KLK6 molecule, enhances KLK6 secretion from the cells. Deactivation of CAV-1, through SRC-mediated phosphorylation, decreased KLK6 secretion. We also demonstrate that, in colon cancer cells, CAV-1 increased the amount of phosphorylated AKT in cells by inhibiting the activity of the AKT-negative regulators PP1 and PP2A. This study demonstrates that proteins such as CAV-1 and AKT, which are known to be altered in colon cancer, affect KLK6 expression and KLK6 secretion.

Intracellular Ca 2+ contributes to K +-induced increase in renal kallikrein secretion

We have reported that natriuretic effects of K + are involved in enhancement of renal kallikrein–kinin system. The study was aimed to examine 1) comparison of augmentative effects of K + on urinary KK excretion with non-specific washout effects by trichlormethiazide (thiazide), polyethyleneglycol 200 (PEG) and rapid physiological saline infusion, 2) contribution of Ca 2+ on the K +-induced increase in renal kallikrein secretion. Renal kallikrein activities were measured as fluorescence activities of methylcoumarinylamide-labeled synthetic substrate of tissue kallikrein (TK). Increases in urinary TK excretion were simultaneously observed with diuresis caused by thiazide, PEG, and rapid saline infusion. K + infusion increased urinary TK excretion with a diuretic response same as the control. K +, but not thiazide, showed an early increase in renal TK secretion dose dependently in the kidney slices. Increases in renal TK secretion persisted during treatment with K +. Neither voltage-dependent Ca 2+-channel blockers such as verapamil and nifedipine nor simultaneous treatment of EDTA affected on the K +-induced increase in renal TK secretion. While, EDTA decreased the K +-induced increases in renal TK secretion with time. Caffeine also had an early effect on the increase in renal TK secretion. K +-induced increases in renal TK secretion was demonstrated even after treatment with ryanodine or depletion of caffeine-sensitive intracellular Ca 2+ by thapsigargin. It was indicated that the increase in renal TK secretion by K + depends on the intracellular Ca 2+ and the caffeine-sensitive release of intracellular Ca 2+ may not be involved in this response. Mechanisms for the K +-induced increase in renal TK secretion needs to be further elucidated.

Overexpression of kallikrein 10 (hK10) in uterine serous papillary carcinomas

Kallikrein 10 is a secreted serine protease recently implicated in the growth and invasion of several human tumors. The goal of this study was to investigate the expression and secretion levels in vitro and in vivo of kallikrein 10 in uterine serous papillary carcinoma, a highly aggressive variant of endometrial tumor. Human kallikrein 10 gene expression levels were evaluated in 11 snap-frozen uterine serous papillary carcinoma biopsies and 6 normal endometrial cell biopsies by real-time polymerase chain reaction. Secretion of kallikrein 10 protein by 10 primary tumor cultures including 3 uterine serous papillary carcinomas, 2 endometrioid carcinomas, and 5 ovarian serous papillary tumors was measured using a sensitive ELISA. Finally, kallikrein 10 concentration in 75 serum and plasma samples from 22 healthy women, 20 women with benign diseases, 21 women with endometrioid carcinomas, and 12 uterine serous papillary carcinoma patients was studied. Kallikrein 10 gene expression levels were significantly higher in uterine serous papillary carcinoma when compared with normal endometrial cell biopsies (mean copy number by real time polymerase chain reaction = 743 versus 1.4; uterine serous papillary carcinoma versus endometrioid carcinoma: P < .02). In vitro kallikrein 10 secretion was detected in all primary uterine serous papillary carcinoma cell lines tested (mean = 2.7 microg/L), and the secretion levels were not significantly different to those found in primary ovarian serous papillary tumor cultures (mean 4.2 microg/L). In contrast, no kallikrein 10 secretion was detectable in primary endometrioid carcinomas. Kallikrein 10 serum and plasma concentrations (microg/L; mean +/- SEM) among normal healthy females (0.6 +/- 0.04), patients with benign diseases (0.6 +/- 0.06), and patients with endometrioid carcinomas (0.7 +/- 0.06) were not significantly different. In contrast, serum and plasma kallikrein 10 values in uterine serous papillary carcinoma patients (1.2 +/- 0.1) were significantly higher than those in the non-cancer group (P = .002), benign group (P = .002), and endometrioid carcinoma patients (P = .005). Kallikrein 10 is highly expressed in uterine serous papillary carcinoma, and it is released in the plasma and serum of uterine serous papillary carcinoma patients. Kallikrein 10 may represent a novel biomarker for uterine serous papillary carcinoma.

A Missing Link Between a High Salt Intake and Blood Pressure Increase

It is widely accepted that a high sodium intake triggers blood pressure rise. However, only one-third of the normotensive subjects were reported to show salt-sensitivity in their blood pressure. Many factors have been proposed as causes of salt-sensitive hypertension, but none of them provides a satisfactory explanation. We propose, on the basis of accumulated data, that the reduced activity of the kallikrein-kinin system in the kidney may provide this link. Renal kallikrein is secreted by the distal connecting tubular cells and all kallikrein-kinin system components are distributed along the collecting ducts in the distal nephron. Bradykinin generated is immediately destroyed by carboxypeptidase Y-like exopeptidase and neutral endopeptidase, both quite independent from the kininases in plasma, such as angiotensin converting enzyme. The salt-sensitivity of the blood pressure depends largely upon ethnicity and potassium intake. Interestingly, potassium and ATP-sensitive potassium (K(ATP)) channel blockers accelerate renal kallikrein secretion and suppress blood pressure rises in animal hypertension models. Measurement of urinary kallikrein may become necessary in salt-sensitive normotensive and hypertensive subjects. Furthermore, pharmaceutical development of renal kallikrein releasers, such as K(ATP) channel blockers, and renal kininase inhibitors, such as ebelactone B, may lead to the development of novel antihypertensive drugs.

Dietary sodium and blood pressure.

The importance of dietary sodium chloride in the regulation of blood pressure has received much attention over the past few years. This area of research and knowledge has been controversial for several reasons. The major reason for controversy is that the science of the relationship is difficult to ascertain. Population science is limited by the narrow range of dietary sodium intake by most populations and individuals and the multiple confounders in those populations at the extremes of intake. Results from clinical trials have been difficult to interpret because of the difficulty in maintaining a given level of sodium intake over a period of time sufficient for study. Furthermore, basic science studies have been challenged by identifying appropriate models that mimic salt-sensitive hypertension in humans. Moreover, a lack of a well-funded proponent in industry complicates the research challenges. Guyton’s concept that the role of the kidney in handling sodium is key to the long-term regulation of blood pressure is now generally accepted.1 However, the exact role that dietary sodium plays in this relationship remains controversial. The relationship between renal handling of sodium and blood pressure is apparently influenced by a complex combination of factors including nutritional, other environmental, genetic, neurohormonal, and metabolic factors. The skepticism of many regarding the role of dietary sodium in blood pressure regulation and control has been tempered by the results of the DASH sodium study.2 Most acknowledge that this study reliably confirmed the benefit of dietary sodium restriction in blood pressure management. The study showed a dose-dependent impact of dietary sodium restriction on blood pressure in older hypertensive and nonhypertensive subjects. For many interested in this area of research, the questions have shifted as a result of the DASH sodium study. Rather than questioning whether dietary sodium is important, many investigators have turned their …

The Renal Kallikrein-Kinin System: Its Role as a Safety Valve for Excess Sodium Intake, and Its Attenuation as a Possible Etiologic Factor in Salt-Sensitive Hypertension

The distal tubules of the kidney express the full set of the components of the kallikreinkinin system, which works independently from the plasma kallikrein-kinin system. Studies on the role of the renal kallikrein-kinin system, using congenitally kininogen-deficient Brown-Norway Katholiek rats and also bradykinin B2 receptor knockout mice, revealed that this system starts to function and to induce natriuresis and diuresis when sodium accumulates in the body as a result of excess sodium intake or aldosterone release, for example, by angiotensin II. Thus, it can be hypothesized that the system works as a safety valve for sodium accumulation. The large numbers of studies on hypertensive animal models and on essential hypertensive patients, particularly those with salt sensitivity, indicate a tendency toward the reduced excretion of urinary kallikrein, although this reduction is modified by potassium intake and impaired renal function. We hypothesize that the reduced excretion of the renal kallikrein may be attributable to a genetic defect of factor(s) in renal kallikrein secretion process and may cause salt-sensitive hypertension after salt intake.Referee: Dr. A.D. Baines, Dept. of Laboratory Medicine & Pathobiology, University of Toronto, 100 College Street, Toronto, ON, Canada, M5G 1L5

Potassium-induced increase in renal kallikrein secretion is attenuated in dissected renal connecting tubules of young spontaneously hypertensive rats

It is suggested that attenuation of the renal kallikrein–kinin system (KKS) involved the development of hypertension in young spontaneously hypertensive rats (SHR). In the present study, a comparison was made between young SHR and Wistar Kyoto rats (WKY) to examine the ability to secrete renal kallikrein from the microdissected connecting tubules (CNT) by potassium or an ATP-sensitive potassium channel blocker, 4-morpholinecarboximidine- N-1-adamantyl- N′-cyclohexylhydrochloride (PNU-37883A), both of which are renal kallikrein secretagogues. Maximum effect of potassium on kallikrein secretion was observed 10 min after placing the tubules at concentration of 20 mM. Kallikrein secretion was also increased concentration-dependently by PNU-37883A (0.1, 1, 10, and 100 μM). In the presence of EDTA, NiCl 2, verapamil, xestspongin C (an inositol 1,4,5-trisphosphate (IP 3) receptor-selective antagonist), or ruthenium red (a ryanodine-sensitive receptor blocker), potassium-induced increase in renal kallikrein secretion was inhibited. Augmentation of renal kallikrein secretion by potassium or PNU-37883A was diminished in SHR compared to WKY. These results indicate that the ability to secrete renal kallikrein by potassium was attenuated in young SHR compared with WKY. Furthermore, it is suggested that the potassium-induced renal kallikrein secretion requires an extracellular Ca 2+ entry through Ca 2+ channels including L-type Ca 2+ channels and Ca 2+ release from intracellular Ca 2+ stores through IP 3 receptor and ryanodine receptor.

Silymarin inhibits function of the androgen receptor by reducing nuclear localization of the receptor in the human prostate cancer cell line LNCaP.

A number of reports have shown that the polyphenolic flavonoid silymarin (SM) is an effective anticancer agent. Agents with novel mechanisms of blocking androgen receptor (AR) function may be useful for prostate cancer prevention and therapy. Previous studies showed that silibinin (SB), the major active component of SM, could inhibit cell proliferation of a human prostate cancer cell line, LNCaP, by arresting the cell cycle at the G(1) phase without causing cell death. This study further delineates the potential molecular mechanism by which SM and SB exhibit antiproliferative effects on androgen-responsive prostate cancer cells by inhibiting function of the AR. We observed that SM and SB inhibited androgen-stimulated cell proliferation as well as androgen-stimulated secretion of both prostate-specific antigen (PSA) and human glandular kallikrein (hK2). Additionally, for the first time, we show that an immunophilin, FKBP51, is androgen regulated and that this up-regulation is suppressed by SM and SB. We further demonstrate that transactivation activity of the AR was diminished by SM and SB using gene transfer of PSA promoter and hK2 androgen-responsive element constructs. However, expression and steroid-binding ability of total AR were not affected by SM in western blotting and ligand-binding assays. Intriguingly, we found that nuclear AR levels are significantly reduced by SM and SB in the presence of androgens using western blotting assay and immunocytochemical staining. This study provides a new insight into how SM and SB negatively modulate androgen action in prostate cancer cells.

Reflex secretion of proteins into submandibular saliva in conscious rats, before and after preganglionic sympathectomy.

1. An indwelling catheter was placed in the left submandibular duct of rats, under pentobarbitone anaesthesia, and connected to an outflow cannula that emerged above the skull. 2. Saliva was collected from the outflow cannula in conscious rats, the same day after recovery from anaesthesia, under four different reflex conditions: grooming, heat exposure, rejection of a bitter tasting substance and feeding on softened chow, repeated in different orders. 3. Saliva flow was greatest for grooming and least for rejection. Protein concentrations were least with heat but much greater and similar for the other stimulations. Acinar peroxidase activity was high for feeding, intermediate for grooming and rejection, and again lowest with heat. Tubular tissue kallikrein activities were moderately low, being greatest with feeding and least with grooming. Secretory immunoglobulin A (SIgA) concentration was least with heat and similar for the other stimulations. 4. The next day, under pentobarbitone anaesthesia, the left preganglionic sympathetic trunk was sectioned (sympathetic decentralization) and, after recovery, the preceding stimulations were repeated. Flow of saliva showed little change, but protein and peroxidase concentrations and outputs decreased dramatically with grooming, rejection and feeding to levels similar to those with heat, which showed little change. Tissue kallikrein was lowered less dramatically, but the reductions in output were significant except with heat. Patterns of proteins resolved by electrophoresis changed for grooming, rejection and feeding and became similar to saliva from heat, which showed little change. No significant effects on SIgA concentrations occurred. 5. Gland weights from the sympathetically decentralized side were greater than from the intact side at the end of the experiments and histologically showed retention of acinar mucin. 6. Thus reflex sympathetic drive varied with the different stimulations; it was least during heat, but it had pronounced effects on acinar secretion of proteins during the other stimulations. At the same time this sympathetic drive had less impact on tissue kallikrein secretion from tubules and had little influence on flow or the concentration of SIgA secreted.

Immunoglobulin a Secretion into Saliva During Dual Sympathetic and Parasympathetic Nerve Stimulation of Rat Submandibular Glands

Salivary secretion of proteins from rat submandibular glands was studied using graded stimulation of the parasympathetic nerve in isolation, and then at a fixed rate in combination with graded sympathetic nerve stimulation. Increasing the frequency of parasympathetic nerve stimulation per se caused a gradual increase in the secretion of peroxidase (from acini) but only small increases in proteinase (from ductal cells) and IgA outputs. Dual stimulations, with an increasing frequency of sympathetic nerve stimulation on a background of low frequency parasympathetic nerve stimulation, showed that maximal acinar secretion of peroxidase required only a low frequency of additional sympathetic stimulation, whereas ductal secretion of kallikrein was greatest with the highest frequency of additional sympathetic stimulation (20 Hz in bursts). IgA secretion also required high frequency additional sympathetic stimulation in bursts for greatest output. Although a synergism occurred with parasympathetic plus sympathetic nerve stimulation for the secretion of both peroxidase and kallikrein it was not evident for the secretion of IgA. This presumably reflects a difference for exocytosis of proteins stored in granules (e.g. peroxidase and kallikrein) compared to those proteins continuously transported across the plasma membrane in vesicles by transcytosis. This work confirms that vesicular movement of secretory IgA can be increased by both parasympathetic and sympathetic nerve stimulation, but the frequency parameters differ for each nerve.

Early increases in renal kallikrein secretion on administration of potassium or ATP-sensitive potassium channel blockers in rats.

1 This study aimed to examine whether administration of potassium or ATP-sensitive potassium channel (KATP channel) blockers caused early increases in renal kallikrein (KK) secretion. To clarify this mechanism, the effect on renal KK secretion of a KATP channel blocker was compared with the effect resulting from use of an osmotic diuretic or volume load. Furthermore, the effect on potassium-induced increases in renal KK secretion by an additional treatment using a KATP channel blocker was examined. Lastly, the effect of a KATP channel blocker on renal KK secretion was also examined in superfused slices of kidney cortex. 2 Intravenous infusion of potassium augmented renal KK secretion within 30 min while urine volume increased gradually in both the potassium loading and control groups. 3 Administration of the KATP channel blocker, 4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexylhydr ochloride (PNU-37883A) or glibenclamide, caused a dose-dependent increase in renal KK secretion. 4 The concentration of KK in urine was higher in the PNU-37883A group as compared to the osmotic-diuretic or volume-load group. 5 PNU-37883A had no additive effect on the potassium-induced increase in renal KK secretion. 6 Renal KK secretion increased in slices of kidney cortex incubated with PNU-37883A within 10 min of superfusion. 7 In conclusion, administration of both potassium and KATP channel blockers induced early increases in renal KK secretion in the absence of the washout phenomenon. Potassium loading may have increased renal KK secretion through the same mechanism as the KATP channel blocker.

K+ loading, but not Na+ loading, and blockade of ATP-sensitive K+ channels augment renal kallikrein secretion

This study aimed to examine whether K+ loading or Na+ loading augments renal kallikrein (KK) secretion. It also investigated the effect of blockade of renal ATP-sensitive K+ channels on renal KK secretion. Rats were administered 50 mmol/kg body weight of KCl. Twelve-hour collected urine was measured for urinary excretion of K+ and Na+ and urinary activity of renal KK. Increases in urinary excretion of K+ and Na+ by K+ loading accompanied an increase in renal KK secretion. In another experiment, rats were infused intravenously with a solution of 75 mM K+ and 75 mM Na+, 150 mM Na+ or 300 mM Na+ for 150 min under anesthesia. Urinary KK activity was measured in urine collected every 30 min. Renal KK secretion began to increase within the 30 min infusion of K+ solution and persisted at more elevated levels during the infusion with K+ solution than with Na+ solutions. Furthermore, rats were given intravenous injection of ATP-sensitive K+ channel blocker, either PNU-37883A (4-morpholinecarboximidine-N-1-adamantyl-N′-cyclohexyl) at a concentration of 10 mg/kg or glibenclamide at 30 mg/kg. Renal KK secretion increased 30 min after administration of both PNU-37883A and glibenclamide. In conclusion, it may be that augmentation of renal KK secretion by K+ loading occurred through an increase in urinary K+ excretion followed by the inhibition of K+ transport from ATP-sensitive K+ channels.