Angiotensinergic Innervation of the Human Right Atrium: Implications for Cardiac Reflexes.

BACKGROUND: The right atrium is densely innervated and provides sensory input to important cardiocirculatory reflexes controlling cardiac output and blood pressure. Its angiotensin (Ang) II-expressing innervation may release Ang II as a neuropeptide cotransmitter to modulate reflexes but has not yet been characterized. METHODS: Intraoperative surgical biopsies from human right atria (n = 7) were immunocytologically stained for Ang II, tyrosine hydroxylase (TH), and synaptophysin (SYN). Tissue angiotensins were extracted and quantified by radioimmunoassay. RESULTS: Angiotensinergic fibers were frequent in epicardial nerves and around vessels with variable TH co-localization (none to >50%/bundle). Fibers were also widely distributed between cardiomyocytes and in the endocardium where they were typically nonvaricose, TH/SYN-negative and usually accompanied by varicose catecholaminergic fibers. In the endocardium, some showed large varicosities and were partially TH or SYN-positive. A few endocardial regions showed scattered nonvaricose Ang fibers ending directly between endothelial cells. Occasional clusters of thin varicose terminals co-localizing SYN or TH were located underneath, or protruded into, the endothelium. Endocardial density of Ang and TH-positive fibers was 30-300 vs. 200-450/mm2. Atrial Ang II, III, and I concentrations were 67, 16, and 5 fmol/g (median) while Ang IV and V were mostly undetectable. CONCLUSIONS: The human right atrium harbors an abundant angiotensinergic innervation and a novel potential source of atrial Ang II. Most peripheral fibers were noncatecholaminergic afferents or preterminal vagal efferents and a minority was presumably sympathetic. Neuronal Ang II release from these fibers may modulate cardiac and circulatory reflexes independently from plasma and tissue Ang II sources.

On-plate self-desalting and matrix-free LDI MS analysis of peptides with a surface patterned sample support.

A hydrophobic-hydrophilic-hydrophobic pattern has been produced on the surface of a silicon substrate for selective enrichment, self-desalting, and matrix-free analysis of peptides in a single step. Upon sample application, the sample solution is first confined in a small area by a hydrophobic F-SAM outer area, after which salt contaminants and peptides are selectively enriched in the hydrophilic and hydrophobic areas, respectively. Simultaneously, matrix background noise is significantly reduced or eliminated because of immobilization of matrix molecules. As a result, the detection sensitivity is enhanced 20-fold compared with that obtained using the usual MALDI plate, and interference-free detection is achieved in the low m/z range. In addition, peptide ions can be identified unambiguously in the presence of NH4HCO3 (100 mM), urea (1 M), and NaCl (1 M). When the device was applied to the analysis of BSA digests, the peptide recovery and protein identification confidence were greatly improved.

Miniaturized multichannel electrospray ionization emitters on poly(dimethylsiloxane) microfluidic devices.

A multichannel electrospray ionization (ESI) emitter was fabricated as part of a poly(dimethylsiloxane) (PDMS) microfluidic device using a three-layer photoresist process which also produces a self-alignment system to make a bonding between the top and bottom PDMS parts. The prototype device (2 cm high x 5 cm wide x 5 cm long) had 16-channels (30 microm wide x 50 microm deep) with emitters of 1 mm length and 60 degrees point angle. The PDMS emitter tips enabled interfacing the device to ESI-mass spectrometry; a stable electrospray from the tips was performed with limits of detection under 1 microM for reference peptides (adrenocorticotropic hormone fragment 1-17, angiotensin I and III).

Automated high-throughput infusion ESI-MS with direct coupling to a microtiter plate.

This paper describes the design and application of instrumentation for automated high-throughput infusion ESI-mass spectrometry. The approach, based on a subatmospheric ESI interface, allows sample introduction from a commercially available microtiter plate without the need for a separate fluid delivery system. The microtiter plate was placed vertically on a three-dimensional translation stage in front of the sampling ESI interface. A single, 7-cm, 20-microm-i.d. fused-silica capillary (total volume, 70 nL), with a tapered tip, served as a combination of sample delivery and spraying capillary. The tapered tip of the capillary was enclosed in a subatmospheric chamber attached in front of the orifice of the mass spectrometer. The sample aspiration rate (flow rate) was regulated by computer-controlled pneumatic valves, which allowed fast switching of the pressure in the subatmospheric ESI chamber. A flow-through wash device was positioned between the microtiter plate and the ESI interface. This design allowed alternate filling of the capillary with (a) sample from the wells and (b) wash solution from the wash device. Sample turnaround times of 10 s/sample, with a 120-nL sample consumption/analysis, and a duty cycle (percentage of total analysis time spent acquiring data) of 40% were achieved. The infusion system was demonstrated in the analysis of preparative HPLC fractions from a small molecule combinatorial library.

A novel derivatization method for peptides to increase sensitivity and backbone fragmentation in liquid secondary-ion mass spectra.

Derivatization is used to increase both negative-ion sensitivity and positive-ion sequence information in the liquid secondary-ion mass spectra (LSIMS) of a series of peptides. The derivatization method involves acylation with pentafluorobenzoyl fluoride in a single-step reaction, and the reaction mixture is applied directly to the probe tip for analysis. Acylation takes place at the unprotected N-terminus, tyrosine, and lysine. The derivatives exhibit increased signal-to-noise ratio for [M-H]- ions, especially where there is not already an acidic amino acid residue in the peptide. In positive-ion LSIMS, the N-terminal group acts to retain the charge at the N-terminus, simplifying the fragmentation by producing N-terminal fragment ions. It also increases positive-ion fragmentation, sometimes very dramatically, making sequence determination more straightforward. The simplicity of the process, together with the enhancements it provides, make this a generally useful method for obtaining peptide structural information.

Tandem mass spectrometry of peptides using hybrid and four-sector instruments: a comparative study.

Product-ion spectra produced by high- and low-energy collisionally activated dissociation (CAD) of [M + H]+ ions of a series of peptides (Mr 550-2500) have been compared on four-sector and hybrid tandem mass spectrometers, respectively. The fast atom bombardment product-ion spectra obtained for the smallest peptide analyzed (methionine-enkephalin) were remarkably similar, but substantial differences in fragmentation were observed for the heavier analytes. For peptides with Mr greater than 1000, more complete sequence information was obtained from high-energy CAD on the four-sector instrument. Nevertheless, low-energy CAD on the hybrid mass spectrometer was able to produce partial sequence information even for the largest of the peptides compared. Limits of analysis, defined as the least quantities of analyte for which product-ion spectra of essentially uncompromised quality could be obtained, were similar (ca. 15 pmol) for small peptides, but lower limits were achieved for larger peptides (Mr greater than 1000) with the four-sector instrument. High-energy CAD spectra were found to be highly reproducible, with qualitatively similar spectra obtained over a wide range of operating conditions. In contrast, it was necessary to carefully control collision gas pressures and collision energies in order to obtain good reproducible data for low-energy CAD. Experimental procedures for obtaining reproducible spectra with good sensitivity for peptides on the hybrid instrument are presented.

Endogenous angiotensin concentrations in specific intrarenal fluid compartments of the rat.

To examine angiotensin (ANG) concentrations in fluid compartments near known intrarenal ANG receptors, we measured ANG concentrations in glomerular filtrate (GF), star vessel plasma (SVP), and luminal fluid from the early, mid, and late proximal tubule (E, M, and L PT). Samples were collected from euvolemic Munich-Wistar rats by free-flow micropuncture; ANG concentrations were measured by RIA. In one group of rats, concentrations of total immunoreactive ANG (reflecting ANG II and lesser amounts of three fragments) in GF and E, M, and L PT fluid averaged 29-40 nM compared with 32 pM in systemic plasma. In a second group, immunoreactive ANG concentrations in SVP also exceeded systemic levels by a factor of 1,000. In a final group, samples of GF and LPT fluid were purified by HPLC before RIA to measure ANG II and III concentrations specifically: their respective concentrations were 6-8 nM and 14-25 nM. We interpret these results to indicate that substantial amounts of ANG peptides are released into or generated within intrarenal fluid compartments, in which local ANG is likely to effect regulation of renal function independently of systemic ANG.

Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland.

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.

Collisionally activated decomposition of modified peptides using a tandem hybrid instrument.

Analyses are described of small peptides and related compounds using a tandem hybrid mass spectrometer of BEQQ geometry. Collisionally activated decomposition of [M + H]+ ions, generated by fast atom bombardment, was performed in the radio frequency (rf)-only quadrupole. Interpretation of fragmentation was greatly facilitated by analysis of labeled analogs, obtained by 18O exchange of carboxyl oxygens. N-Acetylation was also valuable although significant changes in fragmentation resulted from derivatization. Daughter ion spectra of [M + H]+ ions of angiotensin III showed diagnostic fragmentations throughout the peptide chain.

Angiotensin II/III and substance P in human follicular fluid obtained during IVF: relation of the peptide content with follicular size.

Ovarian follicular fluid (FF) of a number of species contain regulatory peptides secreted by granulosa cells or by autonomic nerve terminals. In this report we demonstrate the presence of authentic (HPLC-verification) angiotensin II and III as well as of substance P (SP) in human FF obtained from hMG stimulated infertile patients undergoing in vitro fertilization. Angiotensin II/III (AII/III), estradiol (E2) and progesterone concentrations increase with the size of the follicles. SP concentrations did not vary significantly in FF of various sizes. These peptide concentrations in FF are about 10-fold higher than those measured in the serum of the same patients. Attempts to correlate SP, AII/III, E2 and progesterone concentrations in the individual FF with the ability of an oocyte to be fertilized, failed. Neither AII/III, SP, E2 nor progesterone concentrations were different in these subclasses of FF. Follicles of patients punctured under general anesthesia contained significantly more SP than follicles of patients which had lumbar analgesia. AII/III concentrations were the same in FF of both treatment groups. The presence of angiotensin II and III in FF in increasing concentrations depending on the maturity of the follicle and the inability of general anesthesia to affect the AII/III concentrations suggests that this peptide is produced within the ovary.

The hypothalamic-angiotensin system: location and functional considerations.

Improved immunohistochemical and quantitative microiontophoretic methods were used to characterise angiotensinergic and angiotensin-sensitive neurones in the paraventricular nucleus (PVN) of the rat. The results can be summarised as follows: 1) Angiotensinogen was found in PVN neurones, astrocytes in the diencephalon which make putative contacts with microvessels, and in cells of the choroid plexus. 2) Affinity-purified angiotensin II/III antibodies were used to locate immunoreactive AII/III in large PVN neurones and their fibre tracts which project either caudally or ventrally to the neurohypophysis. 3) Quantitative microiontophoretic studies showed that PVN neurones are more sensitive to angiotensin II than to angiotensin II. 4) Iontophoretic co-application of the selective aminopeptidase inhibitors bestatin and amastatin, together with angiotensin II and angiotensin III produced results consistent with a central role for angiotensin III.

The ovarian renin-angiotensin system: renin-like activity and angiotensin II/III immunoreactivity in gonadotropin-stimulated and unstimulated human follicular fluid.

Renin-like activity and angiotensin II/III immunoreactivity in follicular fluids from 34 women stimulated with human menopausal gonadotropin and human chorionic gonadotropin (56.8 +/- 6.5 ng angiotensin I per milliliter per hour and 187 +/- 21 pg/ml [mean +/- SEM], respectively) were much higher (p less than 0.001) than in follicular fluids from 12 unstimulated preovulatory women (1.41 +/- 0.37 ng angiotensin I per milliliter per hour and 58.5 +/- 13.7 pg/ml) and in simultaneously drawn plasma (4.47 +/- 0.73 ng angiotensin I per milliliter per hour and 31.8 +/- 11.6 pg/ml, respectively; p less than 0.001). Plasma renin-like activity and angiotensin II/III immunoreactivity in stimulated cycles did not differ from unstimulated cycles. Follicular fluid angiotensin II/III immunoreactivity correlated significantly with follicular fluid renin-like activity in stimulated (r = 0.72; p less than 0.01) and in unstimulated samples (r = 0.86; p less than 0.01). Significant correlation was found also between follicular fluid renin-like activity and estradiol. A sharp preovulatory rise of renin-like activity and angiotensin II/III immunoreactivity was noted in unstimulated follicular fluid samples collected on cycle days 13 and 14 compared to days 9 through 12 (p less than 0.01). The findings that follicular fluid renin-like activity and angiotensin II/III immunoreactivity are correlated, and that gonadotropins have a stimulatory effect on follicular fluid concentrations support our concept of a physiologic intrinsic ovarian renin-angiotensin system.

Evidence for the existence of des-Asp1-angiotensin II in human uterine and adrenal tissues.

Renin is present in various tissues outside the kidney. In contrast, the levels of angiotensins (ANG), the active products of the renin-angiotensin system, have not been thoroughly evaluated in tissues. In this study, we demonstrated the presence of immunoreactive (ir) ANG I and ANG II in various human tissues by RIA. Of the tissues examined, uterine tissue contained the most ir-ANG II. Since the anti-ANG II antibody used had significant cross-reactivity with ANG III, high performance liquid chromatography was performed to separate ANG II from ANG III. The major portion of the ir-ANG II in the plasma was ANG II. In contrast, the major portion of the ir-ANG II in uterine tissue was determined to be ANG III, a known biologically active peptide. The adrenal gland and testis also contained ANG III. From these results, it can be postulated that ANG III may contribute to the biological activity of ANG in some tissues.

Characterization of angiotensin I, II, and III from mouse as position-5 isoleucine [Ile5] angiotensins. An HPLC Study.

Angiotensin I was generated in vitro in mouse plasma by addition of mouse submaxillary renin. After deproteinization on Dowex 50W-X2 ion exchange resin, the samples were separated by reverse phase liquid chromatography and the fractions analysed by radio-immunoassay for angiotensin I. Angiotensin I from mouse was injected into the jugular vein of a nephrectomized rat in order to generate angiotensin II and III of mouse origin. Carotid arterial blood was collected immediately, centrifuged and the plasma deproteinized and separated on high pressure liquid chromatography (HPLC). The fractions were analysed for angiotensin I, II and III by radioimmunoassays. Retention times for mouse angiotensins in the liquid chromatography system were compared to synthetic [Val5] and [Ile5] angiotensin I, II and III. All three mouse angiotensins coincided with the corresponding synthetic [Ile5] angiotensins during elution, suggesting the same type of angiotensin in mouse and man. An additional study on rat angiotensins confirmed previous findings by other investigators, indicating that the angiotensins of this animal were also of the [Ile5] type.

A simplified radioimmunoassay for physiologically active angiotensin peptides [(1-8) octa- and (2-8) heptapeptides].

The availability of a sensitive and highly specific rabbit antiserum and the development of a peptide-extraction method employing glass beads permitted the evolution of a rapid reliable radioimmunoassay that measures the sum of the concentration of angiotensin II and its active metabolite, angiotensin III. At a dilution of 1:32,000 the antiserum is capable of measuring 1 fmol (1 pg) of angiotensin II. Cross reactivities of this antiserum, taking angiotensin II as 1.0, are: angiotensin III, 0.75; angiotensin-(3-8) hexapeptide, 0.11; angiotensin I, 0.006; angiotensin-(1-14) tetradecapeptide, 0.0001. The recovery of angiotensin II added to hormone-free plasma was 73 +/- 2% [mean +/- standard deviation (SD), n = 20]. When 0.9 ml of plasma was extracted, the minimal concentration of angiotensin II and III that could be quantified was 4 fmol/ml. When larger volumes of plasma were extracted, sensitivity was enhanced. Plasma blanks were zero. Intra-assay variability was 7.6% SD and interassay variability was 11.7% SD. Angiotensin II and III concentration in venous plasma of normal volunteers on an ad libitum diet was 15 +/- 8 fmol/ml (mean +/- SD, range less than 4 to 35 fmol/ml). The plasma of a patient with primary aldosteronism had an unmeasurable value (less than 4 fmol/ml). Posture, converting enzyme inhibition, and renal artery stenosis resulted in expected changes of angiotensin concentration.

Direct monitoring of sequential enzymatic hydrolysis of peptides by thermospray mass spectrometry.

A procedure for peptide sequencing using an immobilized exopeptidase column directly coupled to a thermospray mass spectrometer is described. The amino acids sequentially released from the C-terminus of the peptide chain are directly introduced into a thermospray ion source by a flowing aqueous buffer. The buffer is essential for the direct production of ions from solution. The method eliminates the need to derivatize the amino acids for detection and, by comparison to standard injections, amino acid sequence information can be obtained in less than two minutes. With the present configuration, detection limits are typically in the low picomolar range.

Generation of angiotensins in cultured pheochromocytoma cells.

Cultured rat pheochromocytoma cells, PC-12, were found to contain renin-like activity. The activity which was inhibited by monospecific antibodies to rat renal renin showed a pH optimum between 6 and 7, indicating that it was a specific renin. The cells also contained angiotensin I and angiotensin I converting enzyme. Angiotensin II/III were also detected in lysate. These findings indicated an intrinsic pathway of angiotensin formation in pheochromocytoma. The formation of angiotensins in pheochromocytoma cells suggests modulating functions of angiotensin on neurotransmitter release from adrenal medulla.

Juxtaglomerular cells grown as monolayer cell culture contain renin, angiotensin I-converting enzyme, and angiotensin I and II/III.

A monolayer cell culture of juxtaglomerular cells (JGC) was derived from the renal cortex of neonatal rats. The JGC had the characteristics of those within the kidney, including peripheral dense bodies and myofibrils indicating a smooth muscle origin; rough ER containing fluffy material consistent with protein synthesis; a prominent Golgi apparatus for packaging granules, and granules having the characteristics of secretory granules and lysosomes. Transplants of the cultured cells into syngeneic recipients survived for 2 weeks or longer and retained the features of JGC. The JGC granules fluoresced when treated with a rabbit antibody against pure rat renin, followed by fluorescein isothyocyanate conjugated F(ab')2 fragment of goat antirabbit IgG (Fc fragment) heavy chain specific. The latter indicated the presence of renin. The JGC were lysed in the presence of DFP, captopril, leupeptin, and EDTA, and were extracted in the presence of pepstatin. The lysate contained renin activity that was inhibited by a specific renin antibody. Nonspecific proteases were excluded by the antibody and its pH optimum. Angiotensin I-converting enzyme was detected in the lysate prepared without the use of EDTA and captopril. Angiotensins I and II/III were derived from the extract by additional extractions, TLC, and RIA, using highly specific antibodies. The angiotensins were confirmed by chromatography monitored by authentic angiotensins. We concluded that the cultured JGC contained renin, angiotensin I-converting enzyme, and angiotensin I and II/III.