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Peptides Derived from the Prohormone ProNPQ/Spexin are Potent Central Modulators of

Cardiovascular and Renal Function and Nociception

Lawrence Toll1, Taline V. Khroyan1, Kemal Sonmez2, Akihiko Ozawa3, Iris Lindberg3, Jay P.

McLaughlin4, Shainnel O. Eans4, Amir A Shahien5, and Daniel R Kapusta5

1. SRI International 333 Ravenswood Ave. Menlo Park, CA 94025. 2. Biomedical Engineering

Department, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Rd., Portland,

Oregon 97239 3. Department of Anatomy and Neurobiology, University of Maryland School of

Medicine, 20 Penn St. Baltimore, MD 21201. 4. Torrey Pines Institute for Molecular Studies,

11350 SW Village Parkway, Port St. Lucie, FL 34987. 5. Department of Pharmacology and

Experimental Therapeutics, Louisiana State University Health Sciences Center-New Orleans,

LA.

Corresponding Author, current address:

Lawrence Toll

Torrey Pines Institute for Molecular Studies

11350 SW Village Parkway,

Port St. Lucie, FL 34987

Tel: 1-772-345-4714

E-mail: ltoll@tpims.org

Running title: Biological Activity of NPQ/spexin

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ABSTRACT

Computational methods have led two groups to predict the endogenous presence of a highly

conserved, amidated, 14-amino acid neuropeptide called either spexin or NPQ. NPQ/spexin is

part of a larger prohormone that contains three sets of RR residues, suggesting that it could

yield more than one bioactive peptide; yet no in vivo activity has been demonstrated for any

peptide processed from this precursor. Here we demonstrate biological activity for two peptides

present within proNPQ/spexin. NPQ/spexin (NWTPQAMLYLKGAQ-NH2) and NPQ53-70

(FISDQSRRKDLSDRPLPE) have differing renal and cardiovascular effects when administered

intracerebroventricularly or intravenously into rats. Intracerebroventricular injection of

NPQ/spexin produced a 13±2 mm Hg increase in mean arterial pressure, a 38±8 bpm decrease

in heart rate, and a profound decrease in urine flow rate. Intracerebroventricular administration

of NPQ53-70 produced a 26±9 bpm decrease in heart rate with no change in mean arterial

pressure, and a marked increase in urine flow rate. Intraventricular NPQ/spexin and NPQ53-70

also produced antinociceptive activity in the warm water tail-withdrawal assay in mice (ED50 <

30 nmol and 10 nmol for NPQ/spexin and NPQ53-70 respectively). We conclude that newly

identified peptides derived from the NPQ/spexin precursor contribute to CNS-mediated control

of arterial blood pressure and salt and water balance, and modulate nociceptive responses.

Key words: antinociception, cardiovascular, renal, neuropeptide

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INTRODUCTION

Recently, computational methods have joined classical pharmacology, reverse

pharmacology (1-3), and chemical methods (4) for the identification of neuropeptides.

Computational methods have been attempted mostly based upon the characteristics of the

prohormones from which active neuropeptides are processed. Particularly important are the

presence and location of the basic residues used for recognition by the prohormone

convertases, which are required to produce the mature and biologically active peptides

(reviewed in (5)). Using different Hidden Markov Model (HMM)-based computational methods,

two independent groups identified a prohormone containing an amidated 14-amino acid peptide

that was respectively called spexin (6) and NPQ (7, 8). This peptide, NWTPQAMLYLKGAQ-

NH2, is identical in humans and mice, and is highly conserved in vertebrate evolution (6). It is,

however, not conserved in the rat, where the carboxy terminal Gly-Arg-Arg, which represents

the putative processing and amidation site, is mutated to Gly-His-Arg.

Locations of both the prohormone and this peptide have recently been determined. The

distribution of preproNPQ/spexin mRNA was examined in human tissues by Northern analysis

by Sonmez et al. who showed expression in several tissues including brain and pancreas, with

highest expression in kidney, suggesting a possible role in salt and water balance (7). In situ

hybridization studies found that in the brain, preproNPQ/spexin mRNA is highly localized in

Barrington’s nucleus, with lesser amounts in the ventrolateral periaqueductal gray. In situ

hybridization studies by Mirabeau demonstrated preproNPQ/spexin mRNA in the stomach (6).

Mirabeau et al, demonstrated NPQ/spexin-induced contractions of the rat stomach fundus

smooth muscle, providing the first known biological activity for this peptide (6). Comprehensive

immunohistochemical studies have recently been performed to determine the localization of

NPQ/spexin immunoreactivity in rat brain and other tissues, using antibodies to the amidated

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human/mouse peptide (9, 10). These studies showed considerable NPQ/spexin staining in skin,

respiratory, digestive, urinary, and reproductive systems, retina, adrenal gland, and various

brain regions.

Due to its high conservation among species and evidence for biological activity,

NPQ/spexin sequence is the most likely peptide derived from the prohormone. However, the

precursor protein contains four RR sequences that could represent potential prohormone

convertase processing sites. Therefore, after cleavage at the end of the signal sequence,

proNPQ/spexin has the potential of generating at least 3 additional biologically active peptides

(see Figure 1), all of which are predicted by Neurpred software (cleavage probability 0.88 for

each RR; http://neuroproteomics.scs.illinois.edu/cgi-bin/neuropred.py). In this study, we show

that both NPQ/spexin and a second novel 18-amino acid peptide, NPQ 53-70

(FISDQSRRKDLSDRPLPE), each identified by our computational HMM, have biological activity

following administration into the central nervous system (CNS) and periphery. Both peptides

evoke marked, but differential, effects on cardiovascular and renal function in conscious rats,

and both peptides demonstrate antinociceptive activity in mice.

MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 275-300 g and

C57Bl/6J mice (Jackson Laboratories, Bar Harbor, ME) or ICR mice (Charles River, Hollister,

CA, USA) weighing 20-25 grams at the start of the experiments were maintained on a 12 h

light/dark cycle (lights on at 7 am) with free access to a normal sodium diet and tap water ad

libitum. Rats were housed 2/group whereas mice were housed 4/group. Rats were used for

cardiovascular and renal function studies, and mice were used for behavioral assessments. All

procedures were conducted in accordance with the National Institutes of Health guidelines for

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the Care and Use of Animals (2002) and were approved by the Institutional Animal Care and

Use Committees.

Peptides. NPQ/spexin and NPQ 53-70 were synthesized by Gen/Script Corp.

Piscataway, NY, USA.

Cardiovascular and renal function studies

Surgical and Experimental Methods: For studies in which peptides or vehicle were

administered into the brain, a stainless steel cannula was stereotaxically implanted into the right

lateral cerebral ventricle of rats anesthetized with ketamine in combination with xylazine, 5-7

days prior to experimentation as previously described (11, 12). On the day of the study, rats

were anesthetized with sodium methohexital (75 mg/kg i.p. and supplemented with 10 mg/kg

given intravenously (i.v.) as needed; JHP Pharmaceuticals, LLC, Rochester, MI) and implanted

with left femoral artery (blood pressure measurement), vein (isotonic saline infusion), and

urinary bladder (urine collection) catheters using standard techniques described previously (10,

11). Following surgical preparation, rats were placed in a rat holder, a chamber with plexiglass

ends connected by stainless steel rods where the metal rods formed an inverted U shape and a

flat base in which the rat sits. This rat holder permits forward and backward movement of the

rat while minimizing movement during surgical recovery and allows for collection of urine. An

i.v. infusion of isotonic saline (55 μl/min) was started and continued for the duration of the

experiment. The experiment commenced after the animal regained full consciousness, and

cardiovascular and renal excretory functions stabilized (4–6 h). Mean arterial pressure and

heart rate were continuously recorded using computer-driven BIOPAC data acquisition software

(MP100 and AcqKnowledge version 3.8.2). Urine volume was determined gravimetrically.

Urine sodium concentration was measured by flame photometry (model 943; Instrumentation

Laboratories, Lexington, MA, USA) and expressed as urinary sodium excretion.

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Intracerebroventricular (i.c.v.) studies in rats: Studies were performed in conscious rats to

determine the cardiovascular and renal responses produced by the administration of

NPQ/spexin or NPQ 53-70 into the brain. Following stabilization of parameters, systemic

cardiovascular function and urine flow rate were measured during a 20-min baseline control

period. Following collection of baseline control measurements, NPQ/spexin (30 nmol), NPQ 53-

70 (30 nmol), angiotensin II (200 ng) or isotonic saline (vehicle; 5 µl) was then administered

i.c.v. Immediately following i.c.v. drug/vehicle administration, experimental urine samples were

collected every 10-min over a 90-min session.

I.V. bolus studies in rats: These studies examined the changes in cardiovascular and

renal excretory function produced by the i.v. bolus injection of NPQ/spexin or NPQ 53-70 in

conscious rats. Cardiovascular parameters and urine flow rate were initially measured during a

20-min baseline period. Next, NPQ/spexin (30, 100, 300 nmol/kg), NPQ 53-70 (30, 100, 300

nmol/kg) or isotonic saline vehicle (200 µl) was injected as an i.v. bolus to conscious rats

(N=6/group). Immediately following administration of the i.v. bolus, experimental urine samples

were collected every 10-min over a 60-min period.

Locomotor activity and antinociceptive studies in mice

Injection techniques. I.c.v. injections into mice were given according to the modified method

of Haley and McCormick (13). Mice were anesthetized with isoflurane and an incision was made

to expose the scalp. Peptides were then injected directly into the ventricle with a 10 μl Hamilton

microsyringe. The coordinates for i.c.v. injection (5 μl) were 2 mm lateral and 2 mm caudal with

respect to bregma, and - 3 mm ventral from the skull surface.

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Antinociceptive testing: the 55oC warm-water tail-withdrawal assay. The nociceptive

stimulus was 55oC water, with the latency to withdraw the tail taken as the endpoint (14, 15).

After determining baseline latencies (1.40 ± 0.03 s), mice (N=7-8/group) received an i.c.v. dose

of vehicle (30% DMSO/70% sterile saline (0.9% NaCl)) or peptide ligand (0.1-30 nmol

NPQ/spexin, 0.1-10nmol NPQ 53-70), with or without a 20 min naloxone pretreatment (10

mg/kg s.c.). Mice were then tested for antinociception every 10-20 min up to 150 min post-

injection. A cut-off time of 15 s was used in this study; if the mouse failed to display a tail-

withdrawal response during that time, the tail was removed from the water and the animal was

assigned a maximal antinociceptive score of 100%. At each time point, antinociception was

calculated according to the following formula: % antinociception = 100 × (test latency – control

latency)/(15 – control latency).

CLAMS measurement of locomotor activity. Locomotor activity was recorded using the

automated, computer-controlled Comprehensive Lab Animal Monitoring System (CLAMS)

apparatus (Columbus Instruments, Columbus, OH, USA; (16)). The cages were 23.5 cm

(Length) x 11.5 cm (Width) x 13 cm (Height) and equipped with infrared emitters along the

longitudinal axis. Locomotor activity was calculated as consecutive beam breakages. Mice

were placed in the cages and habituated for a 45-min period. Animals (N=7-8/group) then

received i.c.v. injections of vehicle (30% DMSO/70% sterile saline, 0.9%), NPQ/spexin (30

nmol, i.c.v.) or NPQ 53-70 (10 nmol, i.c.v.). Doses of NPQ/spexin and NPQ 53-70 were selected

for testing as these doses produced maximal effects in antinociceptive testing in mice.

Following administration of the compounds, mice were returned to test cages for 90 min and

locomotor activity was measured in 60 s intervals.

Statistical analysis. All data are expressed as mean ± SEM. Differences occurring

between treatment groups (e.g. NPQ/spexin peptide vs. vehicle) were assessed by a two-way

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repeated measure ANOVA (SigmaStat Statistical Software, SYSTAT Software, Inc. Point

Richmond, CA, USA for cardiovascular and renal experiments and Prism 5.0 GraphPad

Software, La Jolla, CA, USA for behavioral assessments) with treatment group as the between

group variable and test time as the repeated measure. The magnitude of the changes in

cardiovascular and renal excretory parameters at different time points after i.c.v. or i.v. bolus

injection of NPQ/spexin, NPQ 53-70, or vehicle were compared by a one-way repeated-

measures (ANOVA) with subsequent Dunnett’s test. Post hoc analysis was performed using

Holm-Sidak tests and, where appropriate, a Student’s t- test was also used. Statistical

significance was defined as probability (P) < 0.05.

In vitro proteolysis reactions using proprotein and prohormone convertases

The preparation of mouse PC1/3, PC2 and soluble human furin from Chinese hamster

ovary cell-conditioned medium has been described previously (17-19). The purity of

recombinant enzymes was estimated as greater than 99% using SDS-PAGE stained with

Coomassie brilliant blue (CBB). Two μg of recombinant His-tagged human proNPQ/spexin

were incubated with 2 units of either PC1/3, PC2 or furin in 50 μl reactions containing 100 mM

sodium acetate, pH 5.5 for PC1/3 (for PC2, pH 5.0; for furin, 100 mM Hepes, pH 7.0 was used)

and 5 mM CaCl2 at 37°C for the time periods indicated. The reactions were then subjected to

SDS-PAGE using 18% Tris-HCl acrylamide gels and the gels were stained with CBB. One unit

of PC activity is equal to the amount of the enzyme that is required to cleave 1 pmol/min of the

pRTKR-aminomethyl coumarin (Peptides Int., Lexington, KY, USA) fluorogenic substrate. In

order to determine the cleavage products generated by PC2, 2 μg of His-tagged human

proNPQ/spexin were cleaved by PC2 for 6h at 37°C in 1 ml reactions using the reaction

conditions described above. The cleavage products were applied to a Sep-pak C18 cartridge

(Waters, Milford, MA), and eluted with 60% isopropanol containing 0.1% trifluoroacetic acid, and

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the eluent was lyophilized. The lyophilized sample was resuspended in 2% acetonitrile, 0.1%

trifluoroacetic acid at a concentration of 0.13 mg/ml, and subjected to MALDI-ToF mass

spectrometry.

RESULTS

Cardiovascular and Renal Effects:

As shown in Figure 2, i.c.v. injection of NPQ/spexin (30 nmol) produced an immediate

increase in mean arterial pressure (Fig. 2; control, 121±3 mmHg; NPQ/spexin 10-min, 134±3

mmHg) and decrease in heart rate (control, 386±11 bpm; NPQ/spexin 10-min, 349±11 bpm) in

rats (see Supplemental Fig. S1 for original blood pressure/heart rate tracing). When compared

between treatment groups, there was a significant interaction of drug and time for both mean

arterial pressure (F9,90 = 5.179, p < 0.001) and heart rate (F9,90 = 6.20, p < 0.001). As compared

to respective pre-drug control levels, the pressor and bradycardic responses produced by

central NPQ/spexin remained significantly elevated for 30- and 40-min post-drug injection,

respectively. Concurrent with these cardiovascular responses, i.c.v. NPQ/spexin also produced

a profound and immediate decrease in urine flow rate (control, 57±2 µl/min; NPQ/spexin 10-min,

33±6 µl/min) and a delayed increase in urinary sodium excretion (control, 9.9±1.2 µeq/min;

NPQ/spexin 30-min, 14.4±2.9 µeq/min) (Fig. 2). Differences in mean values showed a

significant interaction of drug and time for urine flow rate (F9,90 = 2.21, p = 0.028) and urinary

sodium excretion (F9,90 = 3.42, p = 0.001). For comparative purposes, changes in systemic

cardiovascular function and urine flow rate were examined in rats that were administered the

neuropeptide, angiotensin II (200 ng) i.c.v. In these studies, i.c.v. angiotensin II produced

significant increases in arterial blood pressure (control, 120±2 mmHg; angiotensin II 10-min,

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137±3 mmHg), and decreases in heart rate (control, 402±17 bpm; angiotensin II 10-min, 360±15

bpm) and urine flow rate (control, 53±2 µl/min; angiotensin II 10-min, 28±4 µl/min).

The i.v. bolus injection of NPQ/spexin (100 and 300 nmol/kg) to conscious rats produced

marked pressor and bradycardic responses that were immediate in onset but of relatively short

duration (20-min; Fig. 3 and Supplemental Fig. S2 for original tracing). The ANOVA indicated

that there was a significant drug x time interaction for both mean arterial pressure (F12,108 =

23.522,) and heart rate (F12,108 = 9.214). Potentially related to their short duration of action in

the periphery, the NPQ/spexin-evoked cardiovascular responses occurred over time (i.e.,

interaction) without significantly altering urine flow rate (Fig. 3, F12,108 = 1.645) or urinary sodium

excretion (Fig. 3 , F12,108 = 1.124). As depicted in Supplemental Fig. S3, the pressor and

bradycardic effects produced by i.v. bolus NPQ were dose-dependent (MAP, F3,20 = 53.65; HR,

F3,20 = 15.226).

The cardiovascular and renal excretory responses produced by the i.c.v. administration

of NPQ 53-70 in conscious rats are shown in Fig. 4. Following drug injection, NPQ 53-70 failed

to alter mean arterial pressure over the 90-min protocol. However, this peptide produced a

significant decrease in heart rate (control, 409 ±17 bpm; NPQ/spexin 10-min, 385±11 bpm),

which persisted for 20-minutes (F9,90 = 1.993) (see Supplemental Fig. S4 for original tracing).

Central NPQ 53-70 also produced a slow onset, but marked increase in urine flow rate, which

was significantly elevated (F9,90 = 3.197, P=0.002) above the respective control value 40-

minutes post drug administration and remained elevated for the remainder of the 90-min

protocol (control, 54±2 µl/min; NPQ 53-70, 90-min, 114±9 µl/min). Concurrent with the diuretic

response, central NPQ 53-70 also produced a significant (F9,90 = 3.174, P=0.002) and sustained

increase in urinary sodium excretion (control, 7.43±0.72 µeq/min; NPQ 53-70, 90-min,

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11.92±0.69 µeq/min). In contrast to central administration, the i.v. bolus injection of NPQ 53-70

did not alter any cardiovascular or renal excretory parameter when tested at doses of 30, 100 or

300 nmol/kg (Supplemental Figure S5 and S6 for original tracing).

Behavioral Effects.

Both NPQ/spexin and NPQ 53-70 were tested for behavioral activity in mice.

Intracerebroventricular administration of NPQ/spexin (30 nmol, i.c.v.) or NPQ 53-70 (10 nmol,

i.c.v.) resulted in an equivalent number of average ambulations over a 90 min period (6.56±0.92

and 6.63±0.71, respectively). These ambulations were not statistically different to those

observed following administration of vehicle (6.89±0.91; one-way ANOVA, F(2,21)=0.04, p=0.96).

Likewise, the overall ANOVA indicated that there was no significant interaction effect

(F(34,357)=1.37) between treatment with saline, NPQ/spexin or NPQ-53-70 (Supplemental Figure

S7 ).

Intracerebroventricular administration of both NPQ/spexin and NPQ 53-70 into mice

induced dose dependent antinociceptive activity using the warm water tail withdrawal assay. A

dose of 10 nmol NPQ 53-70 produced a maximal antinociceptive response at 30 min with an

effect that lasted up to 2 h (Fig. 5A). An overall ANOVA indicated a significant interaction effect

(F(12,105)=4.05) following treatment with NPQ 53-70. Likewise, NPQ/spexin treatment produced a

dose-dependent antinociception, with an overall ANOVA demonstrating an interaction effect

(F(20,165)=13.3). NPQ/spexin was not maximally effective at 10 nmol, but at 30 nmol produced an

antinociceptive response that lasted for greater than 1 h and a dose of 1 nmol produced 50% of

the maximal effect (MPE) at 60 min (Fig. 5B). These robust effects were not opioid receptor-

mediated, as antinociception produced by either peptide was not inhibited by 10 mg/kg

naloxone (s.c.).

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Processing of preproNPQ/spexin.

We investigated processing of recombinant proNPQ/spexin by incubating the bacterially-

derived purified human His-tagged precursor with purified recombinant convertases. A

Coomassie-stained gel is shown in Figure 6. This figure shows that prohormone convertase 2

(PC2), and to a lesser extent prohormone convertase 1 (PC1) and furin, were able to cleave this

precursor into three to four Coomassie-stained fragments. In order to determine the major

products of prohormone convertase cleavage, mass spectroscopic analysis of parallel reactions

was performed. These data show that fragments corresponding to C-terminally extended

NPQ/spexin, residue N(36) to R(51) and N(36) to R(52) (predicted mass of 1849.9377 and

2006.0388, respectively); and fragments corresponding to C-terminally extended NPQ 53-70,

residue F(53) to R(71) and F(53) to R(72) (predicted masses of 2315.2214 and 2471.3325)

were recovered from PC1/3 and PC2 digests. These data confirm that prohormone convertases

can cleave proNPQ/spexin at residues R(35), R(52), and R(72). A high probability of cleavage is

also indicated by the cleavage prediction program NeuroPred;

(http://neuroproteomics.scs.illinois.edu/cgi-bin/neuropred.py). Mass spectroscopy data, while

supporting the existence of the particular cleavages that generate the peptides studied here, do

not rule out the occurrence of other cleavages, since larger and smaller peptides may be difficult

to detect; in fact, larger potential intermediates are evident in Supplemental Fig. S8 .

DISCUSSION

Neuropeptides bind to GPCRs on neurons distributed throughout the brain and

periphery, as well as throughout target organs, and in so doing modulate virtually all

physiological processes. Although many neuropeptides have been studied extensively, and

their physiological actions are well known, the function(s) of others have yet to be revealed. In

particular, a newly discovered neuropeptide, called both spexin and NPQ by its respective

discoverers, is a peptide with biological activities that have yet to be discovered (6, 7).

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Moreover, the prohormone from which NPQ/spexin is derived has several putative processing

sites and therefore has the potential of generating additional biologically active peptides. Here

we report that both NPQ/spexin and a second newly discovered peptide that we have called

NPQ 53-70 have novel cardiovascular, renal, and CNS-mediated behavioral actions.

Both NPQ/spexin and NPQ 53-70 demonstrated potent biological activities in rodents.

When administered by i.c.v. injection to conscious rats, NPQ/spexin produced significant

cardiovascular and renal actions. Intracerebroventricular administration of NPQ/spexin

produced a marked increase in mean arterial pressure and decrease in heart rate in rats

immediately following drug injection. Concurrent with these cardiovascular responses, central

NPQ/spexin also altered renal excretory function, producing an immediate decrease in urine

output and a delayed natriuresis. The cardiovascular and renal responses to NPQ/spexin were

similar to those found after i.c.v. injection of angiotensin II. The similarity in responses produced

by NPQ/spexin and angiotensin II is of considerable interest, since angiotensin II is a highly

potent and efficacious endogenous vasoconstrictor peptide known to participate in the etiology

of neurogenic hypertension (20). In other studies, we have shown that the inhibitory

neuropeptide nociceptin/orphanin FQ also produces centrally-mediated cardiovascular and

renal responses in conscious rats, with i.c.v. dose-ranges between 5 and 20 nmol (12). In

addition to a CNS site of action, the i.v. bolus injection of NPQ/spexin (30, 100 and 300 nmol/kg)

also produced immediate but short-lived dose-related pressor and bradycardic responses in

conscious rats. When administered as an i.v. bolus, the relatively short duration of action of

NPQ/spexin is most likely due to rapid metabolism of the peptide, which might explain its lack of

effect on renal excretory function.

The novel neuropeptide NPQ 53-70 also evoked significant changes in systemic

cardiovascular and renal excretory function when administered into the CNS of rats. However,

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the pattern of responses produced by i.c.v. administration of NPQ 53-70 did not mimic the

responses produced by centrally administered NPQ/spexin. Central NPQ 53-70 (30 nmol)

evoked a transient decrease in heart rate, but failed to alter mean arterial blood pressure.

Alternatively, the bradycardia produced by i.c.v. NPQ/spexin was associated with a concurrent

increase in mean arterial pressure. Further evidence that these two peptides are likely to have

distinct physiological roles in cardiovascular biology is the finding that central NPQ 53-70 also

produced a marked and sustained increase in urine flow rate and urinary sodium excretion.

This is in contrast to the effect of central NPQ/spexin, which instead evoked a pronounced

antidiuretic response and transient natriuresis. Finally, unlike the i.v. bolus administration of

NPQ/spexin, which caused pronounced changes in systemic cardiovascular function, the i.v.

bolus injection of NPQ 53-70 did not alter systemic cardiovascular (or renal excretory) function.

Our studies provide clear evidence that NPQ/spexin and NPQ 53-70 profoundly affect

cardiovascular and renal function in the rat; however, the particular responses generated by

each peptide are different and most likely depend on the locus of action of the ligand (e.g., brain

or periphery). Further studies are required to determine the neural and/or humoral mechanisms

by which NPQ/spexin and NPQ 53-70 peptides influence blood pressure and the handling of

water/sodium, and to determine the brain site(s) mediating the systemic cardiovascular and

renal responses.

Further studies are also required to identify the NPQ/spexin peptide that is active in the

rat. Including species as distant as fish, rat is the only species that has a change in the

cleavage site, with a sequence of Gly-His-Arg, rather than Gly-Arg-Arg. Although not as

common as protein convertase cleavage of Arg-Arg or Lys-Arg, single basic cleavages are well

known, so PC cleavage of Gly-His-Arg is possible (21). Amidation of this peptide is also in

question. C-terminal histidines are hydrolyzed by carboxypeptidase E, but extremely slowly,

with a reaction rate several orders of magnitude lower than substrates with C-terminal lysines or

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arginines (22, 23). However, since carboxypeptidase E has an enzymatic rate several orders of

magnitude greater than protein convertases (24), His removal and subsequent amidation to

produce NPQ/spexin should theoretically be possible in vivo. Alternatively, the active

NPQ/spexin peptide in rats could be NWTPQAMLYLKGAQGH. If so, this would imply that the

NPQ/spexin receptor may recognize the amino terminus of the peptide rather than the carboxy

terminus.

Cardiovascular and renal actions of NPQ/spexin and related peptides are not surprising

considering the demonstrated location of preproNPQ/spexin mRNA in rat brain. In situ

hybridization studies demonstrated intense staining in Barrington’s nucleus (the pontine

micturation center) (7). These studies indicated that proNPQ/spexin-containing cells overlap in

their distribution with cells that express corticotrophin-releasing factor (CRF) in Barrington’s

nucleus as well as serotonergic and dopaminergic cells in the ventrolateral periaquiductal gray.

Barrington’s nucleus contains neurons that send polysynaptic projections to the bladder, colon,

spleen and kidney (25, 26). Activation of CRF-containing neurons in the Barrington’s nucleus

have been proposed to inhibit bladder contraction (i.e., inhibit micturation) (27) and play a role in

mediating stress-induced colonic alterations (28). Barrington’s nucleus also receives neuronal

inputs from the forebrain, including the paraventricular nucleus (PVN) (29). Thus, by acting at

multiple brain sites, NPQ/spexin and/or NPQ 53-70 may play an interactive role with CRF in co-

coordinating gastrointestinal and urinary function with fluid and cardiovascular homeostasis

under basal conditions and during stress/pathology.

When NPQ/spexin and NPQ 53-70 were injected i.c.v. into mice at doses up to 10 nmol

there are no overt changes in behavior compared to control animals. The mice show no

apparent distress, and there is no effect on locomotor activity (Supplemental figure S7). Indeed,

no differences were observed in other normal behaviors such as grooming, rearing, and sniffing

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following NPQ/spexin administration compared to control mice (Supplemental Table T1).

However, there is a very significant effect on response to noxious stimuli. Both NPQ/spexin and

NPQ 53-70 exhibit antinociceptive activity lasting up to 1-2 h post injection. In the warm water

tail-withdrawal paradigm, NPQ 53-70 is longer lasting and more potent than NPQ/spexin, and

produces a maximal antinociceptive response between 30 and 50 min, at a 10 nmol dose (Fig.

4). Naloxone did not block the antinociceptive activity of either peptide, indicating that this

response is not opioid receptor-mediated. Thus, while NPQ/spexin and NPQ 53-70 showed

differential effects on cardiovascular and renal function, these two peptides were similar in their

CNS ability to evoke antinociception. Taken together these antinociceptive, systemic

cardiovascular, and renal excretory responses produced by NPQ 53-70 are the first

demonstration of biological activity of this peptide, a second peptide product of the

proNPQ/spexin precursor.

Despite the demonstrated biological activity of chemically synthesized NPQ/spexin and

NPQ 53-70, the endogenous presence of these particular peptide species in the brain, or other

organs, has not yet been conclusively demonstrated. Experiments examining the in vitro

hydrolysis of proNPQ/spexin with PC2 have shown that the brain-expressed enzyme PC2 can

efficiently process this precursor and produce the expected basic-residue extended versions of

both NPQ/spexin and NPQ 53-70 (see Supplemental Figure S8), indicating likely endogenous

production of NPQ/spexin and NPQ 53-70. Mirabeau et al have shown that NPQ/spexin-

immunoreactive peptides co-localize with insulin in secretory vesicles (6), suggesting that

proNPQ/spexin-derived peptides possess the correct targeting signals to arrive at the secretory

granule compartment, where PC2 is located.

A great deal remains to be learned about this neuropeptide precursor and its processed

peptides. Experiments designed to identify the molecular forms of NPQ/spexin and NPQ 53-70

17

using immunological methods are currently underway. The receptor(s) mediating the actions of

these two peptides must also be identified. In addition, based upon the proximity of

preproNPQ/spexin mRNA to CRF in Barrington’s nucleus and dopaminergic neurons in the

ventrolateral periaqueductal gray, which project to the CRF-containing area of the bed nucleus

of the stria terminalis, there is reason to believe that NPQ/spexin-derived peptides may have a

significant role in the stress response and potential stress-mediated gastrointestinal

disturbances (7, 30).

Our initial experiments provide significant CNS and peripheral actions for neuropeptides

arising from a single precursor protein highly localized to very limited brain regions. This very

discrete localization is similar to that of two other recently discovered peptides, NPS and

hypocretin/orexin, both of which have a wide range of important physiological functions

emanating from a very small population of neurons in a discrete brain region (31, 32). We

anticipate a similar impact of proNPQ/spexin-derived peptides in health and disease.

18

Acknowledgements: This work was supported by NIH grant R21 DA016629 to LT, American Heart Association grant

0855293E and NIH grant P20 RR018766 to DRK, and NIH grant DA027170 to IL.

19

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23

Figure Legends

Figure 1. Amino acid sequence of ppNPQ/spexin from human, mouse, and rat. Bold letters

show potential processing sites. Areas shaded in gray show NPQ/spexin and NPQ 53-70.

NPQ 53-70 includes an unprocessed RRK.

Figure 2. Effect of i.c.v. NPQ/spexin (30 nmol) and isotonic saline vehicle on heart rate (HR),

mean arterial pressure (MAP), urine flow rate (V) and urinary sodium excretion (UNaV) in rats.

N=6 ±SEM/group; *, p<0.05, vs resp. group control (C); Φ, p<0.05 vs saline value at same time

point.

Figure 3. Time course responses in cardiovascular and renal excretory function produced by

i.v. bolus NPQ/spexin in conscious Sprague-Dawley rats. Abbreviations as in Fig. 1. N=6

±SEM/group; : *,t p<0.05, vs resp. group control (C); Φ, P<0.05 vs saline value at same time

point.

Figure 4. Effect of i.c.v. NPQ 53-70 (30 nmol) and isotonic saline vehicle on heart rate (HR),

mean arterial pressure (MAP), urine flow rate (V) and urinary sodium excretion (UNaV) in rats.

N=6 ±SEM/group, *, p<0.05, vs resp. group control (C); Φ, P<0.05 vs saline value at same time

point.

Figure 5. Antinociceptive activity ±SEM of NPQ 53-70 (A) and NPQ/spexin (B) after i.c.v.

administration, (N=7 or 8/group), to mice in the warm water tail withdrawal procedure.

Figure 6. A Coomassie-stained gel of proNPQ/spexin produced by incubating the bacterially-

derived purified recombinant proNPQ/spexin precursor with purified recombinant convertase.

Figure  1  

! ! !!!!!!!!!"!!!!!!!!!!!!Signal sequence!!!!!!!!!!!!!!!!!!!!!!!!!#!!!!!!!!!!!!!!!!!!!!!!"!!!!!!!NPQ/Spexin !!!#! 10 20 30 40 50 Homo sapiens MKGLRSLAATTLALFLVFVFLGNSSCAPQRLLERRNWTPQAMLYLKGAQGRR Mus musculus MKGPSVLAVTAVVLLLVLSALENSSGALQRLSEKRNWTPQAMLYLKGAQGRR Rattus norvegicus MKGPSILAVAALALLLVLSVLENSSGAPQRLSEKRNWTPQAMLYLKGAQGHR 60 70 80 90 100 110 Homo sapiens FISDQSRRKDLSDRPLPERRSPNPQLLTIPEAATILLASLQKSPEDEEKNFDQTRFLEDSLLNW Mus musculus FLSDQSRRKELADRPPPERRNPDLELLTLPEAAALFLASLEKSQKGADEGGNFDKSELLEDRLFNW Rattus norvegicus FISDQSRRKELADRPPPERRNPNLQLLTLPEASAAFLASLEKPQKDEGGDFDKSKLLEDRRFYW

[ NPQ- 53-70 ]

Figure  5                                                A                                                      B  

Figure6

Supplemental Fig. S1. Original tracing of changes in heart rate (HR), arterial blood

pressure (BP), and mean blood pressure (MAP) produced by the i.c.v.

administration of NPQ/spexin (30 nmol) in a conscious Sprague-Dawley rat.

Supplemental Fig. S2. Original tracing of changes in heart rate (HR), arterial blood

pressure (BP), and mean blood pressure (MAP) produced by the i.v. bolus

administration of NPQ/spexin (100 nmol/kg) in a conscious Sprague-Dawley rat.

Supplemental Fig. S3. Peak changes in heart rate (HR) and mean arterial pressure

(MAP) produced by i.v. bolus NPQ/spexin in conscious Sprague-Dawley rats. N=6

±SEM/group; *, p<0.05 vs resp. group control (C); ψ, p<0.05 vs. NPQ/spexin 30 nmol/kg

Supplemental Fig. S4. Original tracing of changes in heart rate (HR), arterial blood

pressure (BP), and mean blood pressure (MAP) produced by the i.c.v.

administration of NPQ 53-70 (30 nmol) in a conscious Sprague-Dawley rat.

Supplemental Fig. S5. Peak changes heart rate (HR) and mean arterial pressure

(MAP) produced by i.v. bolus NPQ 53-70 in conscious Sprague-Dawley rats. N=2

±SEM/group.

Supplemental Fig. S6. Original tracing of changes in heart rate (HR), arterial blood

pressure (BP), and mean blood pressure (MAP) produced by the i.v. bolus

administration of NPQ 53-70 (100 nmol/kg) in a conscious Sprague-Dawley rat.

Supplemental Figure S7 . Effect of i.c.v. injection of NPQ/spexin and NPQ 53-70

on locomotor activity.

Supplemental Figure S8 . PC2 digest of ppNPQ/spexin. The digest was performed on human ppNPQ/spexin. Mass spectrometry was performed by the Stanford University PAN facility. Molecular masses shown below were detected and represent extended forms of ppNPQ/spexin peptides.

1849.9377 NWTPQAMLYLKGAQGR; 2006.0388 NWTPQAMLYLKGAQGRR

2315.2214 FISDQSRRKDLSDRPLPER; 2471.3225 FISDQSRRKDLSDRPLPERR

NPQ

NPQ-long

Supplemental Table T1. Effect of NPQ53-70 on normal mouse behaviors. Mice were

injected i.c.v. with 2 μl PBS or NPQ 53-70 (10 nmol). They were then observed for 30 min in an

open field chamber. Episodes of grooming, sniffing, and rearing were counted every 10 s for

each animal during the 30 min test period.

Grooming Sniffing Rearing PBS 17.00 ± 14 36.00 ± 13 9.50 ± 8.5

NPQ 53-70 15.33 ± 6.9 46.00 ± 14.5 10.67 ± 4.8