Frieder Kees

Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal axis and synthesize cortisol

The skin and its major appendages are prominent target organs and potent sources of key players along the classical hypothalamic‐pituitary axis, such as corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and α melanocyte stimulating hormone (α‐MSH), and even express key steroidogenic enzymes. Therefore, it may have established local stress response systems that resemble the hypothalamic‐pituitary‐adrenal (HPA) axis. However, functional evidence that this is indeed the case in normal human skin in situ has still been missing. We show that microdissected, organ‐cultured human scalp hair follicles respond to CRH stimulation by up‐regulating proopiomelanocortin (POMC) transcription and immunoreactivity (IR) for ACTH and α‐MSH, which must have been processed from POMC. CRH, α‐MSH, and ACTH also modulate expression of their cognate receptors (CRH‐R1, MC1‐R, MC2‐R). In addition, the strongest stimulus for adrenal cortisol production, ACTH, also up‐regulates cortisol‐IR in the hair follicles. Isolated human hair follicles secrete substantial levels of cortisol into the culture medium, and this activity is further up‐regulated by CRH. CRH also modulates important functional hair growth parameters in vitro (hair shaft elongation, catagen induction, hair keratinocyte proliferation, melanin production). Finally, human hair follicles display HPA axis‐like regulatory feedback systems, since the glucocorticoid receptor agonist hydrocortisone down‐regulates follicular CRH expression. Thus, even in the absence of endocrine, neural, or vascular systemic connections, normal human scalp hair follicles directly respond to CRH stimulation in a strikingly similar manner to what is seen in the classical HPA axis, including synthesis and secretion of cortisol and activation of prototypic neuroendocrine feedback loops.

Determination of macrolides in biological matrices by high-performance liquid chromatography with electrochemical detection

A liquid chromatographic method for the determination of macrolide antibiotics is described using a cyanopropyl column which proved to be as efficient or superior to the normally used apolar reversed-phase columns. The recovery of the macrolides from water and plasma was 80-90%. Using 0.5 ml of plasma, 30 ng/ml of clarithromycin, 50 ng/ml of roxithromycin and 10 ng/ml of azithromycin could be determined with acceptable precision and accuracy. The method has been employed in pharmacokinetic studies in humans for the determination of roxithromycin, clarithromycin and azithromycin in plasma, serum and other biological matrices. The particular selectivity of the cyanopropyl phase may also allow the simultaneous determination of erythromycin and its prodrug esters.

Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of acetylsalicylic acid (ASA) and its main metabolite salicylic acid (SA) in human plasma. Acidified plasma is deproteinized with acetonitrile which is separated from the aqueous layer by adding sodium chloride. ASA and SA are extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: Novapak C18 4 microns silica, 150 x 4 mm I.D.; eluent: 740 ml water, 900 microliters 85% orthophosphoric acid, 180 ml acetonitrile) and photometric detection (237 nm). 2-Methylbenzoic acid is used as internal standard. The method allows the determination of ASA and SA in human plasma as low as 100 ng/ml with good precision (better than 10%). The assay was used to determine the pharmacokinetic parameters of ASA and SA following oral administration of 100-500 mg ASA in healthy volunteers.

Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice

Hypertension is an underlying risk factor for cardiovascular disease. Despite this, its pathogenesis remains unknown in most cases. Recently, the transient receptor potential (TRP) channel family was associated with the development of several cardiovascular diseases linked to hypertension. The melastatin TRP channels TRPM4 and TRPM5 have distinct properties within the TRP channel family: they form nonselective cation channels activated by intracellular calcium ions. Here we report the identification of TRPM4 proteins in endothelial cells, heart, kidney, and chromaffin cells from the adrenal gland, suggesting that they have a role in the cardiovascular system. Consistent with this hypothesis, Trpm4 gene deletion in mice altered long-term regulation of blood pressure toward hypertensive levels. No changes in locomotor activity, renin-angiotensin system function, electrolyte and fluid balance, vascular contractility, and cardiac contractility under basal conditions were observed. By contrast, inhibition of ganglionic transmission with either hexamethonium or prazosin abolished the difference in blood pressure between Trpm4-/- and wild-type mice. Strikingly, plasma epinephrine concentration as well as urinary excretion of catecholamine metabolites were substantially elevated in Trpm4-/- mice. In freshly isolated chromaffin cells, lack of TRPM4 was shown to cause markedly more acetylcholine-induced exocytotic release events, while neither cytosolic calcium concentration, size, nor density of vesicles were different. We therefore conclude that TRPM4 proteins limit catecholamine release from chromaffin cells and that this contributes to increased sympathetic tone and hypertension.

Cytokines down-regulate α1-adrenergic receptor expression during endotoxemia

ObjectiveThe reduced pressure response to norepinephrine in septic patients has directed our interest to the regulation of &agr;1-adrenergic receptors in vitro and in vivo during conditions mimicking acute sepsis. DesignProspective animal trial followed by a controlled cell culture study. SettingLaboratory of the Department of Anesthesiology. SubjectsMale Sprague-Dawley rats weighing 200 to 250 g and a mesangial cell line. InterventionsExperimental endotoxemia was induced in rats with lipopolysaccharide, and blood pressure dose-response studies with norepinephrine were performed. &agr;1-Receptor gene expression was determined in various organs by a specific RNase protection assay, and tissue concentrations of the proinflammatory cytokines interleukin-1&bgr; and tumor necrosis factor-&agr; were measured. Rat renal mesangial cells were incubated with these cytokines or with nitric oxide donors to investigate the regulation of &agr;1-adrenergic receptors during severe inflammation on a cellular level. Measurements and Main ResultsThe pressor effect of norepinephrine was markedly diminished during endotoxemia. The animals showed down-regulated mRNA levels of &agr;1A-, &agr;1B- and &agr;1D-receptors in all organs investigated, and the tissue concentrations of interleukin-1&bgr; and tumor necrosis factor-&agr; were highly increased during experimental endotoxemia. Incubation of cultured rat renal mesangial cells with the cytokines resulted in diminished &agr;1B-receptor gene expression and [H]prazosin binding capacity, whereas incubation of the cells with nitric oxide donors did not affect &agr;1B-receptor expression. In line, blocking of cytokine-induced nitric oxide synthesis by coincubation of mesangial cells with NG-nitro-l-arginine methyl ester did not influence cytokine-induced down-regulation of &agr;1B-receptors. ConclusionsOur data show that endotoxemia causes a systemic down-regulation of &agr;1-receptors on the level of gene expression and suggest that this effect is likely mediated by proinflammatory cytokines in a synergistic but nitric oxide-independent fashion. We propose that this down-regulation of &agr;1-adrenergic receptors contributes to the attenuated blood pressure response to norepinephrine and, therefore, to septic circulatory failure in patients.

Via β-adrenoceptors, stimulation of extrasplenic sympathetic nerve fibers inhibits lipopolysaccharide-induced TNF secretion in perfused rat spleen

Using a spleen slice microsuperfusion technique in mice, we have previously characterized the role of norepinephrine (NE) and other neurotransmitters for sympathetic modulation of IL-6 and TNF secretion of splenic macrophages. Since experiments in spleen slices do not reflect the situation of an entire perfused organ, we investigated sympathetic modulation of lipopolysaccharide (LPS)-induced secretion of IL-6 and TNF in perfusion experiments of rat spleen. In an organ bath, perfusion was performed in explanted whole spleens, and catecholamines and cytokines were measured by HPLC and ELISA, respectively. Release of NE depended on stimulation frequency (maximum at 10 Hz). Apart from NE, perfusates also contained significant amounts of dopamine and epinephrine. Furthermore, perfusate epinephrine levels correlated positively with perfusate NE levels (RRank=0.750, p<0.001) but not with plasma epinephrine concentrations. This indicates that epinephrine is a conversion product of sympathetically released NE. Early electrical stimulation of extrasplenic splenic nerves, 20 min after administration of LPS, significantly inhibited TNF secretion. This electrically induced effect was abrogated by simultaneous administration of propranolol (10(-6) M) but it was not influenced by administration of either an alpha1- or alpha2-adrenergic antagonist. Late electrical stimulation of splenic nerves, 2.5 h after administration of LPS, did not change TNF secretion. Interestingly, no influence of early or late sympathetic nerve fiber stimulation on IL-6 secretion was observed. In conclusion, this is the first perfusion study of the entire spleen that demonstrates that early electrical stimulation of sympathetic splenic nerve fibers directly inhibits LPS-induced TNF secretion. This study corroborates the idea that splenic sympathetic nerves are able to inhibit important activators of the innate immune system when stimulation happens very early or even prior to their induction by LPS.

Bioavailability of Mycophenolate Mofetil and Enteric‐Coated Mycophenolate Sodium Is Differentially Affected by Pantoprazole in Healthy Volunteers

The influence of pantoprazole 40 mg twice daily on the bioavailability of a single dose of mycophenolate mofetil 1000 mg or enteric‐coated mycophenolate sodium is investigated in healthy volunteers. The plasma concentrations of mycophenolic acid and of the inactive metabolite mycophenolic acid glucuronide are measured by high‐performance liquid chromatography. The pharmacokinetic parameters following sole administration are similar for mycophenolate mofetil and enteric‐coated mycophenolate sodium except for the time to peak concentration, which is longer in the enteric‐coated mycophenolate sodium group. Concomitant treatment with pantoprazole significantly (P < .001) lowers the mycophenolic acid exposure following administration of mycophenolate mofetil. The peak concentrations drop by 57%, and area under the curve decreases from 0 to 12 hours by 27%. In contrast, pantoprazole does not change the pharmacokinetics of enteric‐coated mycophenolate sodium. Given that mycophenolic acid exposure correlates with the incidence of biopsy‐proven acute rejections in renal transplant recipients, these findings may have clinical implications. Administration of pantoprazole in combination with mycophenolate mofetil could possibly result in an insufficient mycophenolic acid exposure, increasing the risk of treatment failure.

Low sodium and furosemide-induced stimulation of the renin system in man is mediated by cyclooxygenase 2.

The aim of this study was to examine the effects of highly selective inhibition of cyclooxygenase 2 (COX‐2) with rofecoxib on the renin system during long‐term stimulation and after short‐term stimulation. Six healthy male volunteers received, in a randomized crossover design, a low‐sodium diet for days 1 through 9 with or without 25 mg rofecoxib twice daily on days 5 through 9 and, in addition, 20 mg of furosemide intravenously on day 8. Plasma renin activity increased 2 to 3 times over baseline with a low‐sodium diet and 5 times over baseline 30 minutes after intravenous furosemide; it was still elevated nearly 5 times on day 9. These effects were completely blocked by rofecoxib. Plasma aldosterone and urinary aldosterone concentrations basically reflected the findings with plasma renin activity. Urinary sodium excretion decreased during a low‐sodium diet and increased after intravenous furosemide without being significantly affected by rofecoxib. We have concluded that low‐sodium and furosemide‐stimulated renin and aldosterone secretion is completely blocked in healthy volunteers during COX‐2 inhibition with rofecoxib, suggesting that intact COX‐2 is of major importance for stimulation of the renin system under these conditions in man.

Cyclic cytidine 3′,5′-monophosphate (cCMP) signals via cGMP kinase I

We analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane‐permeable cCMP analogue dibutyryl‐cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP‐dependent protein kinase (cGK) Iα and Iβ and stimulated cGK in aorta lysates. cCMP‐induced relaxation was abolished in cGKI‐knockout tissue. Additionally, deletion of inositol–trisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP‐mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP‐mediated inhibition of platelet aggregation was absent in cGKI‐ and IRAG‐deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.

Brief report: Pharmacokinetics of ciprofloxacin in young (healthy volunteers) and elderly patients, and concentrations in prostatic fluid, seminal fluid, and prostatic adenoma tissue following intravenous administration

C iprofloxacin has a broad antibacterial spectrum with favorable pharmacokinetic properties and can be administered orally as well as parenterally. Therefore, it has been used widely in the treatment of complicated nosocomial urinary tract infections and demonstrated to be suitable for therapy of bacterial prostatitis and for perioperative prophylaxis in urologic surgery. In this study, the pharmacokinetics of ciprofloxacin after intravenous administration in younger volunteers and elderly patients were compared, and concentrations in prostatic and seminal fluid and in prostatic adenoma tissue following intravenous administration were determined.