Neuropeptide Y is a 36-amino-acid peptide CO-stored with noradrenaline in sympathetic nerves supplying the cardiovascular system (1). Neuropeptide Y contracts human blood vessels in vitro (2) and in vivo (3). In conscious rats, systemic administration of NPY is 6 times more potent than noradrenaline and produces a long-lasting increase in blood pressure because of a marked increase in total peripheral resistance (4).
The previous absence of specific competitive NPY-receptor antagonists has made receptor characterization difficult to perform (5). However, by using modified NPY analogs, at least three receptor subtypes have been suggested; Y1, Y2 and Y3 [for review see (6)]. Bioactivity as well as binding studies indicate that the vascular NPY-receptor is mainly of the Y1 type (7). However, there is also evidence for Y2-mediated vasoconstriction [e.g., in the splenic vascular bed of the pig (8)].
We previously demonstrated that NPY elicits powerful contractile effects in small human subcutaneous (s.c.) arteries and characterized the receptor mediating the contraction by using antisense oligodeoxynucleotides (9). Furthermore, by using reverse transcriptase-polymerase chain reaction (RT-PCR), we have shown messenger RNA (mRNA), corresponding to a sequence of the human NPY Y1-receptor to be present in smooth-muscle cells cultivated from human s.c. arteries (10-12). In our study, the recently developed selective NPY Y1-receptor antagonist BIBP3226, modified NPY analogs, and RT-PCR were used to characterize the NPY receptors in human s.c. resistance arteries. The aim was to characterize the receptors mediating the contractile effects of NPY in arteries that might be of relevance for blood-pressure regulation in human beings.
MATERIALS AND METHODS
Isolation of total RNA. Human s.c. resistance arteries were frozen in liquid nitrogen, and total cellular RNA was extracted by using the TRIzol Reagent (GIBCO BRL, Life Technologies, Sweden) (13). Frozen tissues were homogenized with 1 ml of TRIzol Reagent and RNA further extracted with chloroform. RNA was precipitated from the aqueous phase by the addition of isopropanol. The RNA pellet was finally washed with 70% ice-cold ethanol, air-dried, dissolved in 20 μl of diethylpyrocarbonate-treated water and stored at -20°C until use.
The purity and amount of total RNA was determined spectrophotometrically by measurement of optical density of an aliquot at 260 nm and 280 nm. The ratio of absorption (260:280 nm) of all preparations was between 1.6 and 1.8. Finally, samples were subjected to gel electrophoresis and stained with ethidium bromide to prove the integrity of the 18 and 28S ribosomal RNA.
To eliminate DNA contamination, total RNA was pretreated with Amplification Grade DNase I (GIBCO BRL) in 1× DNase I reaction buffer, in the presence of 20 units of RNase inhibitor (Perkin Elmer, Sweden). To inactivate the DNase I, 1 μl of 25 mM EDTA solution (pH 8.0) was added to each tube, and samples were heated for 10 min at 65°C.
Reverse transcriptase-polymerase chain reaction (RT-PCR). Synthesis of first strand cDNA and subsequent PCR was carried out by using the GeneAmp RNA PCR kit (Perkin-Elmer) in a PCR DNA Thermal Cycler (Perkin-Elmer).
DNase-treated RNA samples were reverse transcribed to complementary DNA (cDNA) in a 20-μl reaction volume in the presence of 1 ′ PCR buffer (50 mM KCl, 10 mM Tris-HCl; pH 8.3), 5 mM MgCl2, 1 mM of each deoxyribonucleoside triphosphate (dNTP), 50 pmol of random hexamers, and 50 units of M-MLV (Moloney murine leukemia virus) reverse transcriptase. The samples (20 μl) were incubated at room temperature for 10 min, at 42°C for 15 min, and then inactivated at 99°C for 5 min and immediately cooled on ice.
The PCR amplification reaction was composed of 20 μl of the first-strand cDNA reaction mixture and 80 μl master mix containing 1′ PCR buffer, 2 mM MgCl2, 50 pmol each of NPY Y1-receptor mRNA specific forward and reverse primers, and 2.5 units of AmpliTaq DNA polymerase. The reaction mixture was subjected to 35 cycles of PCR amplification. After an initial denaturation step at 95°C for 2 min, the cycle profile included denaturation for 1 min at 95°C and annealing for 1 min at 60°C. After the final cycle, the temperature was maintained at 72°C for 7 min to allow completion of synthesis of amplified products. As a negative control of the PCR reaction, some tubes omitted the RT enzyme in the first-strand cDNA reaction of total RNA.
Electrophoretic analysis. Ten microliters from each PCR product were electrophoresed on a 1% agarose gel (GIBCO BRL), containing 1.0 μg/ml ethidium bromide (Sigma E 1510), in 1 × TBE buffer (89 mM TRIS-borate, 2 mM EDTA; pH 8.0) at 5 V/cm for 1.5 h. This analysis was performed in a 6.5 × 10 cm Minicell, Model EC 370 (E-C Apparatus Corporation; Techtum Lab AB, Sweden). A 100-base pair (bp) DNA ladder (Promega, SDS, Sweden) was run in one of the outside lanes to confirm molecular size of the amplification product.
Oligonucleotide primers. Oligonucleotide primers based on published cDNA nucleotide sequences from the human NPY Y1 receptor (12) were used in the PCR to amplify a 520-bp fragment corresponding to a region spanning the transmembrane domain 4 and 7 of the human NPY Y1-receptor mRNA sequences. The following primers were used; NPY Y1 forward; 5′-TATGTAGGTATTGCTGTGATTTG-3′, corresponding to nucleotides 661-683 and NPY Y1 reverse; and 5′-CTGGAAGTTTTTGTTCAGGAAC/TCCA-3′, corresponding to nucleotides 1156-1180. These two oligonucleotide primers were obtained from Scandinavian Gene Synthesis AB, Sweden.
In vitro pharmacology
S.c. resistance arteries from 18 patients who underwent surgery for nonvascular diseases (e.g., hernia, gallbladder disease, or colon tumors) were dissected out at the beginning of the operation from the abdominal region and cut into cylindrical segments (2-3 mm long) with intact endothelium. The segments were suspended in a double-mantled small volume (2.5 ml) tissue bath made of Perspex. Two small stainless steel wires (diameter, 100 μm) were gently inserted into the vessel lumen under a microscope. The thin wires were supported by relatively thick metal tubes, one of which was connected to a force-displacement transducer (FT03C) attached to a Macintosh plus computer and the other to a displacement device. The position of the opposite holder could be moved be means of a movable unit, allowing fine adjustments of vascular tension by varying the distance between the steel wires. The experiments were continuously recorded by the Macintosh software program Chart. The mounted specimens were immersed in temperature-controlled (37°C) tissue baths containing a buffer solution of the following composition (in mM/L): NaCl, 119; NaHCO3, 15; KCl, 4.6; MgCl, 1.2; NaH2PO4, 1.2; CaCl2, 1.5; and glucose, 11. The solution was continuously gassed with 5% CO2 in O2, giving a pH of 7.4. A tension of 4 mN was applied to the vessel segments, and they were allowed to stabilize at this level of tension for 1.5 h. The contractile capacity of each vessel segment was examined by exposure to a potassium-rich (60 mM) buffer solution (14). After another 45-min rest period, agonists were added to the vessels in cumulative doses. The antagonist was present for 15 min before addition of agonists. All antagonist experiments were performed with an internal control (i.e., neuropeptide Y alone added to a segment of the same blood vessel). Maximal contraction in percentage of 60 mM KCl is expressed as Emax. The potency of the agonists is expressed as pEC50 (the negative logarithm of the molar concentration of the agonist that produces 50% of the maximal effect). The potency of the antagonist is expressed as pA2 (the negative logarithm of the molar concentration reducing the effect of the agonist to half of the original effect) (15,16). Values represent mean ± SEM, and n refers to the number of patients from whom the vessels segments were collected. Statistical significant differences were determined with Wilcoxon sign-rank test by using StatView II on a Macintosh IIcx. A p < 0.05 was considered significant. The study was approved by the Ethics Committee of Lund University.
Drugs. The following drugs were used: neuropeptide Y (Auspep, Australia), NPY13-36, NPY18-36 and PYY (Bissendorf Biochemicals), Pro34NPY (a kind gift from Thue Schwartz, Copenhagen), noradrenaline and α,β-methylene adenosine triphosphate (ATP) (Sigma) and BIBP3226 (a generous gift by Drs. H. Doods and K. Rudolf, Dr. Karl Thomae GmbH, Germany). To avoid adhesion to glass materials, the peptides were dissolved in saline containing 10 mg/ml bovine serum albumin (BSA).
Human s.c. resistance arteries with a diameter of 0.2-0.4 mm were used for experiments shortly (within 6 h) after surgical dissection. The potassium contractions were 3.0 ± 0.64 mN. NPY, PYY, and Pro34NPY all induced powerful contractions with equal potency (Fig. 1). NPY13-36 and NPY18-36 had no contractile effects at all on the vessels tested ≤3 μM. BIBP3226 had no contractile effect by itself in concentrations ≤1 μM but inhibited potently the NPY-induced vasoconstriction. With increasing concentrations of BIBP3226, the concentration-response curve for NPY was moved to the right without change of the maximum contractile response (Fig. 2A). The pEC50 values were significantly reduced (Table 1). Schild plot analysis of BIBP3226 revealed a pA2 of 8.53 ± 0.22 and a slope of 0.99 ± 0.14 that did not differ from unity (Fig. 2B). When using a nonlinear regression for analysis of competitive agonist-antagonist interactions, we revealed a pKb of 8.27 ± 0.10, which was not significantly different from the pA2 value received with Schild-plot analysis.
In concentrations ≤1 μM, BIBP3226 did not affect the contractile responses of noradrenaline or α,β-methylene ATP in concentrations ≤100 μM (data not shown). The presence of RT-PCR products of the expected size (520 bp) corresponding to mRNA encoding the human NPY Y1 receptor and an additional band ≈100 bp longer were detected in human s.c. resistance arteries from two patients and presented on an agarose gel (Fig. 3). False-positive RT-PCR results seem unlikely because contamination of DNA was eliminated by treating all RNA samples with RNase-free DNase. Furthermore to determine whether the amplification product came exclusively from RNA, and RT reaction was run in which the enzyme was replaced by RNase-free water.
Our study was an attempt to characterize the receptor subtype responsible for the NPY-induced constriction of human s.c. resistance arteries to evaluate the possible role of NPY in the regulation of peripheral resistance and blood-pressure control.
Earlier experiments with a series of C-terminal peptide fragments of NPY led to the proposal of the existence of at least two different NPY-receptor subtypes; a postsynaptic (Y1) and a presynaptic (Y2) receptor (17). However, there is also evidence for postsynaptic Y2-mediated vasoconstriction [e.g., in the splenic vascular bed of the pig (8)]. The Y2 receptor is characterized by its activation by truncated NPY fragments (e.g., NPY13-36 and Ac-[Leu28, Leu31]-NPY24-36). The Y1 receptor is selectively activated by Pro34NPY, and a third receptor subtype (Y3), is characterized by being insensitive to PYY (6). In our study, the responses to Pro34NPY and PYY did not differ from the contractile effect of NPY either in sensitivity or in maximum effect. Neither NPY13-36 nor NPY18-36 had any contractile effects in the studied concentrations. The effect of Pro34NPY and the lack of contractile effects of NPY13-36 and NPY18-36 indicated the absence of functionally important Y2 receptors in human s.c. resistance arteries. Vasoconstriction mediated by a Y3 receptor seems unlikely because PYY induced as potent and powerful contractions as did NPY.
The absence of specific and competitive NPY antagonists has hampered the detailed pharmacologic characterization of the NPY receptors (5). Recently however, BIBP3226, a selective and highly potent nonpeptide NPY Y1-receptor antagonist, was developed (18). In vitro, BIBP3226 inhibits the NPY-induced contraction of human cerebral arteries (19,20). In vivo in animal models, BIBP3226 antagonizes the NPY-induced increase in blood pressure (ID50 = 0.11 mg/kg, i.v.) with no effect on noradrenaline-, endothelin-, vasopressin-, or angiotensin II-induced pressor responses. NPY antagonism could also be observed in vascular Y1-receptor models such as the perfused rat kidney, mesentery or rabbit ear preparations but not in rat colon, a NPY Y3-receptor bioassay, in which BIBP3226 failed to alter the contractile effect of NPY (21,22). BIBP3226 did not displace [125I]NPY from the human NPY Y2 receptor neuroblastoma cell line (SMS-KAN) (23). With BIBP3226, Lundberg and Modin (24) demonstrated a role for endogenous NPY for the long-lasting duration of sympathetic vasoconstrictor effect in the pig hind limb. BIBP3226 (1 mg/kg, i.v.) caused a 50% reduction of the long-lasting vasoconstrictor response to sympathetic nerve stimulation at high frequency (40 impulses at 20 Hz).
We studied the antagonistic effects of BIBP3226 on the NPY-induced vasoconstriction in human s.c. resistance arteries. BIBP3226 had no vascular effects per se but had potent antagonistic effects on the NPY-induced contraction. Increasing concentrations of the antagonist caused a rightward shift of the concentration-response curve for NPY. Schild-plot analysis indicated a competitive antagonism with a pA2 value of 8.53 ± 0.22. Recently Lew and Angus (16) published a new way of analyzing competitive agonist-antagonist interactions by the use of nonlinear regression. When our data were reanalyzed with this new method, a pKb value of 8.27 ± 0.10 was obtained, which is not significantly different from the pA2 value achieved with the Schild-plot analysis. These results indicate that the contraction in the studied arteries is mediated solely by NPY Y1 receptors and are in agreement with a selective antagonism of BIBP3226 at NPY Y1 receptors.
BIBP3226 might serve as a valuable tool in studying the importance of NPY in sympathetic neurotransmission. Our data demonstrate an antagonistic effect to NPY in low concentrations with no antagonistic effect at the vascular receptors for the sympathetic cotransmitters noradrenaline or ATP in human s.c. resistance arteries. The effect of BIBP3226 on vascular resistance and blood pressure in vivo during different levels of sympathetic activation in human beings is therefore of considerable interest. The plasma half-life of BIBP3226 is only ≈30 min (oral communication of Lundberg and Modin, 1995). However, more stable variants of the molecule may serve as drugs reducing peripheral vascular resistance and blood pressure.
Finally we used RT-PCR and detected mRNA encoding the human NPY Y1 receptor in human s.c. resistance arteries. As has been shown before in human cerebral arteries (20) and smooth muscle cells cultivated from human s.c. arteries (10), an additional product ≈100 bp longer was detected in the s.c. resistance arteries studied. The sequence of the NPY Y1-receptor gene contains a small intron (97 bp) with a stop codon after the fifth transmembrane region (11). The extra product may code for a splice variant of the receptor missing two transmembrane regions. The physiologic significance of this splice variant is unknown.
In conclusion, we showed that BIBP3226 is a potent antagonist of the NPY-induced contraction of human s.c. resistance arteries. The results, together with data on modified NPY analogs, indicates that the contractile effect is mediated by human NPY Y1 receptors. No other NPY-receptor subtype seems to be involved. Furthermore we demonstrated the presence of mRNA encoding the human NPY Y1 receptor in human s.c. resistance arteries. Because NPY is a potent constrictor of human peripheral arteries, it may contribute to the development of hypertension. Knowing that NPY Y1 receptors mediate contraction in human s.c. resistance arteries might be of relevance when selecting future blood pressure-reducing agents.
Acknowledgment: The study was supported by the Swedish Medical Research Council (grant 5958), the Medical Faculty of Lund University, the Royal Physiographic Society, Lund, the Swedish Medical Society, and the Swedish Hypertension Society.
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