Torill Berg

Physiological Regulation of the Secretion of Histatins and Statherins in Human Parotid Saliva

The small salivary phosphoproteins, histatins and statherins, have important functions in the oral cavity in terms of antimicrobial actions and regulation of calcium phosphate homeostasis. Neither the effects of various physiological stimuli on their secretion nor the nature of the efferent receptor involved in the stimulus-secretion coupling has been determined previously. These aspects are important for improved understanding of the secretory control of salivary proteins and may have implications regarding the effects of specific medications on salivary constituents and oral health. The effects of graded mechanical (chewing on short and long silicone tubings) and gustatory stimulation (0.5, 1.5, and 5.0% citric acid) on the secretion of histatins and statherins were studied in the presence and absence of adrenolytic agents (n = 10). In this model, secretory rates of both proteins increased with increases in flow rate, with 5.0% citric acid representing a particularly potent stimulus. Histatin and statherin secretory rates were significantly reduced by the β 1-adrenolytic agent (histatins to 58 to 72% and statherins to 11 to 29% of that in corresponding control experiments), but not by the α 1-adrenolytic agent. Since the β1-adrenergic receptors played an important role in the stimulus-secretion coupling of these proteins, protective salivary functions in the oral cavity may be compromised during β 1-adrenolytic treatment.

Immunohistochemical localization of two angiotensin I-converting isoenzymes in the reproductive tract of the male rabbit.

The male reproductive tract contains two different isoenzymes of angiotensin I-converting enzyme (ACE), i.e., pulmonary and testicular ACE. The present study shows selectively the cellular distribution of the ACE isoenzymes in the reproductive tract of male rabbit, using indirect immunofluorescence or immunoperoxidase methods. Testicular ACE was found in the seminiferous tubules of the testes in spermatocytes containing mature spermatids, and in spermatids within the epididymal tubular lumen in sexually mature, but not in immature, rabbits. Epididymal tubular cells contained pulmonary ACE. In the young rabbit, epididymal tissue contained more ACE than that in adult rabbit, since ACE was observed in principal cells in addition to basal cells. In mature rabbit, ACE was observed in basal cells only. Strong staining for pulmonary ACE was observed in cells of the vas deferens in both young and adult rabbit. Therefore, synthesis of epididymal ACE, unlike the testicular isoenzyme, was not stimulated by sexual maturation. Enzymatically active ACE in seminal fluid corresponds to the pulmonary isoenzyme. The present study indicates that this seminal fluid ACE may originate from cells of the epididymal tubules, particularly those of the vas deferens. Endothelial cells of blood vessels lying in the interstitium of both testicular and epididymal tissue contained the pulmonary isoenzyme.

Role of kinin in regulation of rat submandibular gland blood flow

In tissues rich in kallikrein, vasodilator kinins, acting as paracrine hormones, may play a role in the local regulation of blood flow. We studied the role of kinins in the regulation of blood flow in the rat submandibular gland using a kinin analogue with antagonistic properties, [DArg0]Hyp3-Thi5-8[DPhe7]bradykinin. When infused into the carotid artery (20 micrograms/min/rat), this antagonist blocked the effect of bradykinin (25-250 ng/kg, intracarotid injection) on glandular blood flow. In nephrectomized rats, the antagonist also blocked the increase in glandular blood flow caused by enalaprilat, a kininase II converting enzyme inhibitor. At a dose of 20 micrograms/min/rat, the antagonist produced no detectable change in basal glandular blood flow; however, at a higher dose (100 micrograms/min/rat), it caused a significant decrease (p less than 0.001). In eight of 10 rats, blood flow decreased by 75% or more; this effect was not blocked by the alpha-adrenergic receptor antagonist phentolamine. After antagonist infusion was stopped, blood flow returned toward normal. Sympathetic nerve stimulation of the gland induced vasoconstriction followed by poststimulatory vasodilatation. In rats displaying severe vasoconstriction after the antagonist, postsympathetic vasodilatation was abolished even when stimulation was performed after the antagonist infusion had been stopped and blood flow returned toward normal. Although a direct vasoconstrictor effect of the kinin antagonist cannot be completely ruled out, these data suggest that, in the rat submandibular gland, kinins may play a role in regulation of basal blood flow and vasodilatation after converting enzyme inhibitor or sympathetic stimulation.

Role of β1–3-Adrenoceptors in Blood Pressure Control at Rest and During Tyramine-Induced Norepinephrine Release in Spontaneously Hypertensive Rats

&bgr;-Adrenoceptors contribute to hypertension in spite of the fact that &bgr;-adrenoceptor agonists lower blood pressure. We aimed to differentiate between these functions and to identify differences between spontaneously hypertensive and normotensive rats. &bgr;-Adrenoceptor antagonists with different subtype selectivity or the ability to cross the blood-brain barrier were used to demonstrate &bgr;-adrenoceptor involvement in resting blood pressure and the response to tyramine-induced peripheral norepinephrine release. The centrally acting propranolol (&bgr;1+2[+3]), CGP20712A (&bgr;1), ICI-118551 (&bgr;2), and SR59230A (&bgr;3), as well as peripherally restricted nadolol (&bgr;1+2) and atenolol (&bgr;1), were administered intravenously, separately, or in combinations. Blood pressure, cardiac output, heart rate, total peripheral vascular resistance, and plasma catecholamine concentrations were evaluated. &bgr;-Adrenoceptor antagonists had little effect on cardiovascular baselines in normotensive rats. In hypertensive rats, antagonist-induced hypotension paralleled reductions in resistance, except for atenolol, which reduced cardiac output. The resistance reduction involved primarily neuronal catecholamine, central &bgr;1-adrenoceptors, and peripheral &bgr;2-adrenoceptors. Tyramine induced a transient, prazosin-sensitive vascular resistance increase. Inhibition of nerve-activated, peripheral &bgr;1/3-adrenoceptors enhanced this &agr;1-adrenoceptor–dependent vasoconstriction in normotensive but not hypertensive rats. In hypertensive rats, return to baseline was eliminated after inhibition of the central &bgr;1-adrenoceptor, epinephrine release (acute adrenalectomy), and peripheral &bgr;2/3-adrenoceptors. Adrenalectomy eliminated &bgr;-adrenoceptor–mediated vasodilation in hypertensive rats, and tyramine induced a prazosin-sensitive vasoconstriction, which was inhibited by combined blockade of central &bgr;1- and peripheral &bgr;2-adrenoceptors. In conclusion, nerve-activated &bgr;1- and &bgr;3-adrenoceptor–mediated vasodilation was not present in hypertensive rats, whereas epinephrine-activated &bgr;2- and &bgr;3-adrenoceptor–mediated vasodilation was upregulated. There was also a hypertensive, nerve-activated vasoconstrictory mechanism present in hypertensive rats, involving central &bgr;1- and peripheral &bgr;2-adrenoceptors combined.

The role of nitric oxide, adrenergic activation and kinin‐degradation in blood pressure homeostasis following an acute kinin‐induced hypotension

1 Nitric oxide (NO) has been suggested as the mediator of the vascular response to bradykinin. In the present study, we found that NO did not mediate the hypotensive response to bradykinin. In addition, the significance of kininase II in terminating a kinin‐induced hypotension and the role of the adrenergic system in compensating for the acute fall in blood pressure (BP) was established. 2 In normal rats, the NO‐synthase inhibitor Nco‐nitro‐L‐arginine methyl ester (L‐NAME) induced a rise in basal BP (ΔBP = 40 ± 6 mmHg, P < 0.0014) which was not altered by pretreatment with phentolamine (ΔBP = 50 ± 6 mmHg, NS). L‐NAME did not attenuate the acute fall in BP in response to bradykinin (3–30 μg kg−1) or kallikrein (6–300 μg kg−1). However, a significant decrease was observed in the duration of the hypotensive response (P < 0.027). This shorter duration was not observed after pretreatment with phentolamine in addition to L‐NAME. Phentolamine alone prolonged the hypotensive response to bradykinin (P < 0.04). These experiments confirm the role of NO‐formation as a hypotensive component in BP homeostasis but not the role of NO as a mediator in kinin‐induced hypotension. It further shows that the continuous NO‐release also impedes the compensatory adrenergic hypertensive response following the acute fall in BP induced by bradykinin. 3 The hypertensive respose to intravenously administered phenylephrine was found to be unchanged by preadministration of L‐NAME (NS) thus snowing that L‐NAME did not change the sensitivity to the adrenergic response. In a separate protocol on L‐NAME‐treated rats we found no difference in heart rate (NS) during the recovery period following bradykinin before as compared to after administration of phentolamine. It was therefore concluded that the observed alterations in the duration of the hypotensive response were most probably due to changes in peripheral vascular resistance. 4 To confirm further that NO is not a mediator in kinin‐induced hypotension, we used an experimental model where the response to bradykinin was prolonged by preventing kinin degradation by kininase II‐converting enzyme inhibitor (CEI). To produce a hypotensive response purely dependent on kinin, the studies were performed after removal of the renin‐angiotensin system by nephrectomy (Nx). In this model, bradykinin (6 μg kg−1, i.v.) induced a prolonged hypotensive response. Pretreatment with L‐NAME did not alter the magnitude or the progression of the hypotensive response to bradykinin, thus confirming that NO was not a mediator in BK‐induced hypotension. 5 To study the mechanisms involved in terminating the hypotensive response to bradykinin, the results from the Nx CEI‐treated rats were compared with Nx animals not treated with CEI. In the latter group, bradykinin induced a short hypotensive response, i.e. 0.5 ± 0.1 min as compared to the 17 ± 1 min after CEI (P < 0.003). After kininase II‐inhibition (and L‐NAME), BP recovery was totally dependent on the adrenergic system, since phentolamine prevented a recovery in BP during the experimental period (P > 0.01, compared to the CEI/L‐NAME group). These results demonstrate the importance of kininase II as the major agent in terminating a bradykinin‐induced hypotension, whereas the adrenergic system plays a small, although significant role in compensating for the fall in BP. The continuous release of NO therefore not only lowers basal BP but also impedes the compensatory adrenergic response.

Analysis of the pressor response to the K+ channel inhibitor 4-aminopyridine.

The cardiovascular response to the K(+) channel inhibitor 4-aminopyridine in anaesthetized rats was analysed. 4-Aminopyridine produced a biphasic pressor response. First, it increased blood pressure, total peripheral vascular resistance, cardiac output and stroke volume. Nitric oxide synthase (NOS) inhibitor augmented the tension response; reserpine, phentolamine, propranolol, scopolamine, atropine, adrenalectomy, indomethacin, angiotensin AT(1) and endothelin ET(A) receptor antagonists had no effect. Subsequently, heart rate increased, but total peripheral vascular resistance was no longer elevated. Reserpine and propranolol abolished the tachycardia. An elevated late tension occurred after propranolol and NOS inhibitor but not reserpine or phentolamine+NOS inhibitor. The peripherally acting 3,4-diaminopyridine produced similar responses. 4-Aminopyridine contracted isolated aortic rings also after denudation. These results are compatible with that the immediate tension response resulted from closure of vascular smooth muscle K(+) channels, and that closure of presynaptic K(+) channels in peripheral sympathetic nerves subsequently activated noradrenaline release, beta-adrenoceptors and tachycardia, while nitric oxide counter-acted a concomitant alpha-adrenergic vasoconstriction.

The vascular response to the K+ channel inhibitor 4-aminopyridine in hypertensive rats

The K+ channel inhibitor 4-aminopyridine induced an immediate increase in blood pressure and tension in spontaneously hypertensive rats (SHR). Further analysis strongly suggested this to be due to closure of vascular smooth muscle K+ channels, as previously concluded for normotensive rats (WKY). The tension response was greater in SHR than WKY, suggesting an increased channel activity in order to compensate for the high total peripheral vascular resistance in SHR. The response was enhanced after nitric oxide (NO) synthase inhibitor in both strains, probably reflecting increased channel activity after elimination of the NO-cGMP pathway. The response in SHR but not WKY was increased after alpha(1)-adrenoceptor inhibition and adrenalectomy but not sympathetic nerve transmitter depletion. It increased also after angiotensin AT(1) and endothelin ET(A) receptor antagonists and protein kinase C inhibitor. These results indicated an increased adrenal catecholamine, angiotensin AT(1) and endothelin ET(A) activation of the phospholipase C-protein kinase C pathway in SHR, inhibiting the 4-aminopyridine-sensitive K+ channels.

Processing of Epidermal Growth Factor in the Rat Submandibular Gland by Kallikrein-Like Enzymes

Epidermal growth factor (EGF) is synthesized as a precursor which is processed intracellularly to a 6 kDa EGF in the rat submandibular gland. This gland contains very high amounts of kallikrein-like enzymes, and the purpose of the present study was to examine whether any of five such enzymes, rK1, rK2, rK7, rK9 or rK10, can process the rat EGF precursor. Molecular weight forms of EGF, that were N- or C-terminally extended compared to submandibular gland EGF were obtained from rat urine. These extended forms of EGF were incubated with each of the enzymes for 24 h at 37 degrees C. Two enzymes, rK7 and rK10, were able to cleave N- and C-terminally extended EGF, releasing a form of EGF which eluted similarly to submandibular gland EGF upon gel filtration, and which was recognized both by antibodies against rat EGF and by the EGF receptor. One enzyme, rK1, cleaved C- but not N-terminally extended EGF. Neither rK2, nor rK9 cleaved the extended forms of EGF. In previous immunohistochemical studies rK1, rK7 and rK10 have all been demonstrated in the EGF containing cells of the rat submandibular gland. EGF and rK1 are also synthesized in the rat kidney but the present study demonstrated that EGF and rK1 are not colocalized in this organ. Based on the cleavage of the extended forms of rat EGF by rK1, rK7 and rK10 and on the fact that the enzymes are abundant and colocalized with EGF in the rat submandibular gland, we suggest that rK1, rK7 and rK10 can be involved in the processing of the EGF precursor in the rat submandibular gland.

Simultaneous Parasympathetic and Sympathetic Activation Reveals Altered Autonomic Control of Heart Rate, Vascular Tension, and Epinephrine Release in Anesthetized Hypertensive Rats

Sympathetic hyperactivity and parasympathetic insufficiency characterize blood pressure (BP) control in genetic hypertension. This shift is difficult to investigate in anesthetized rats. Here we present a pharmacological approach to simultaneously provoke sympathetic and parasympathetic transmitter release, and identify their respective roles in the concomitant cardiovascular response. To stimulate transmitter release in anesthetized normotensive (WKY) and spontaneously hypertensive rats (SHR), we injected intravenously 4-aminopyridine (4-AP), a voltage-sensitive K+ channel (KV) inhibitor. A femoral artery catheter monitored BP, an ascending aorta flow-probe recorded cardiac output and heart rate (HR). Total peripheral vascular resistance (TPVR) was calculated. 4-AP-induced an immediate, atropine (muscarinic antagonist)- and hexamethonium (ganglion blocker)-sensitive bradycardia in WKY, and in both strains, a subsequent, sustained tachycardia, and norepinephrine but not epinephrine release. Reserpine (sympatholytic), nadolol (β-adrenoceptor antagonist) or right vagal nerve stimulation eliminated the late tachycardia, adrenalectomy, scopolamine (central muscarinic antagonist) or hexamethonium did not. 4-AP increased TPVR, transiently in WKY but sustained in SHR. Yohimbine (α2-adrenoceptor antagonist) prevented the TPVR down-regulation in WKY. Reserpine and prazosin (α1-adrenoceptor antagonist) eliminated the late vasoconstriction in SHR. Plasma epinephrine overflow increased in nadolol-treated SHR. Through inhibition of KV, 4-AP activated parasympathetic ganglion transmission and peripheral, neuronal norepinephrine release. The sympathetic component dominated the 4-AP–HR-response in SHR. α2-adrenoceptor-dependent vasodilatation opposed norepinephrine-induced α1-adrenergic vasoconstriction in WKY, but not SHR. A βAR-activated, probably vagal afferent mechanism, hampered epinephrine secretion in SHR. Thus, 4-AP activated the autonomic system and exposed mechanisms relevant to hypertensive disease.

Immunohistochemical localization of rat submandibular gland esterase B (homologous to the RSKG-7 kallikrein gene) in relation to other serine proteases of the kallikrein family.

The rat submandibular gland contains several members of the kallikrein family. In the present study we purified and raised an antiserum against one of these enzymes, i.e., esterase B, which was first described by Khullar et al. in 1986. N-terminal amino acid analysis revealed complete homology between esterase B and the kallikrein family gene RSKG-7. For characterization of the antiserum, flat-bed isoelectrofocusing with immunoblotting was superior to immunoelectrophoresis and double immunodiffusion in detecting and identifying crossreacting proteins. This was due to the fact that kallikrein-like enzymes were readily separated by isoelectrofocusing, and immunoreactivity was easily detected by the sensitive peroxidase-anti-peroxidase staining after blotting onto nitrocellulose membrane. Immunohistochemical controls were carried out accordingly, including homologous as well as crossreacting antigens. In the submandibular gland, esterase B was detected exclusively in all granular convoluted tubular cells, co-localized with tissue kallikrein and tonin. Some staining was also observed in striated duct cells; however, this staining reaction was induced by cross-reactivity with kallikrein, since staining was abolished by addition of kallikrein as well as esterase B to the primary antiserum. It was therefore concluded that like tonin and antigen gamma, but unlike kallikrein, esterase B was not detected in the striated ducts of the submandibular, parotid, or sublingual glands. This separation in anatomic distribution between esterase B and kallikrein may indicate that prokallikrein activation is not the only biological function of esterase B.